Recombinant interferon-beta muteins

ABSTRACT

The invention relates to novel interferon-beta activity (IbA) proteins and nucleic acids. The invention further relates to the use of the IbA proteins in the treatment of IFN-β related disorders.

This application is a continuing application of U.S. Ser. No.60/133,785, filed May 12, 1999.

FIELD OF THE INVENTION

The invention relates to novel interferon-beta activity (IbA) proteinsand nucleic acids. The invention further relates to the use of the IbAproteins in the treatment of interferon-beta (INF-β) related disorders.

BACKGROUND OF THE INVENTION

Human Interferons (IFNs) are members of a biologically potent family ofcytokines. Originally, IFNs were identified as agents produced andsecreted by virus-infected cells which can protect cells against furtherviral infections. However, in addition to this antiviral effect, IFNscan elicit many other changes in cellular behavior, including effects oncellular growth and differentiation and modulation of the immune system[e.g., see Lengyel, Annu. Rev. Biochem. 51:251-82 (1982); Gresser andTovey, Biochim. Biophys. Acta 516(2):231-47 (1978); Gresser et al.,Nature New Biol. 231(18):20-1 (1971); Dolei et al., J. Gen. Virol.46(1):227-36 (1980); Gresser, Cell Immunol 34(2):406-15 (1977)]. Byvirtue of their antigenic, biological and physico-chemical properties,IFNs are classified into three groups, INF-α (leukocyte), INF-β(fibroblast) and INF-γ (immune) [Stewart, J. Infect. Dis. 142(4):643(1980)].

In humans, the IFN-α subtype encompass a multigene family of about 20genes, encoding proteins of 166-172 amino acids that are all closelyrelated. In contrast to this diversity, there is only one humaninterferon-beta (IFN-β) gene, also encoding a protein of 166 aminoacids. IFN-β has low homology to the IFN-α family and is an N-linkedglycoprotein [Knight, Proc. Natl. Acad. Sci. U.S.A. 73(2):520-523(1976)]. There is also only one human IFN-γ gene that encodes apolypeptde of 143 amino acids that is glycosylated and forms a dimer inits native state. IFN-γ shows only slight structural similarities toIFN-α or to IFN-β.

All IFN-α and IFN-β (also commonly referred to as type I interferonfamily) appear to bind to a common high affinity cell surface receptor,a 130 kD glycoprotein that is widely distributed on different cell typesand that is distinct from the one bound by IFN-γ. Type-I interferons arerecognized by a complex containing the receptor subunits ifnar1 andifnar2 and their associated Janus tyrosine kinases, Tyk2 and Jak1, thatactivate the transcription factors STAT1 and STAT2, leading to theformation of the transcription factor complex ISGF3[interferon-stimulated gene factor 3; Li et al., Biochemie80(8-9):703-20 (1998); Nadeau et al., J. Biol. Chem. 274(7):4045-52(1999)]. Three distinct modes of IFN/receptor complex interaction areknown: (i) INF-α with ifnar1 and ifnar2; (ii) IFN-β with ifnar1 andifnar2; and (iii) IFN-β with ifnar2 alone [Lewerenz et al., J. Mol.Biol. 282(3):585-99(1998)]. While Lewerenz et al. suggest that INF-α andIFN-β interact with their receptors in different ways and as such mayalso signal differently, the events responsible for biological activitybeyond receptor binding are poorly understood.

As might be predicted for such a large family of cytokines with almostubiquitously distributed receptors, IFNs display varied physiologicalroles. Production of IFN-α or IFN-β is induced by infection, includingviral infection or the presence of foreign cell types and antigens. Itis not clear what specific molecules are responsible for induction, butdouble-stranded RNA and cytokines can be good inducers. There is muchoverlap between different cell types in both the inducers and thespecies of IFN that is induced. The major cell types that produce IFNsare: lymphocytes, monocytes and macrophages (for IFN-α); fibroblasts andsome epithelial cells and lymphoblastoid cells (for IFN-β); andactivated T lymphocytes (for IFN-γ).

In addition to the ‘classical’ anti-viral activities that all IFNselicit in their target cells, the biological consequences of IFN bindingto its receptor can include inhibition of cell proliferation, inductionof cell differentiation, changes in cell morphology, enhancement ofhistocompatibility antigen expression on many cells and stimulation ofimmunoglobulin-Fc receptor expression on macrophages. B lymphocytes canbe induced to increase antibody production by low concentration of IFN-αor IFN-β. An additional effect of IFN-α and IFN-β is activation ofnatural killer cells that may be responsible for the destruction ofvirus-infected cells or tumor cells in vivo. Overall, IFNs seem to be ofgreat importance as part of the body's defense against foreignorganisms, foreign antigens and abnormal cell types (Clemens, inCytokines, BIOS Scientific Publishers Limited, 1991; De Maeyer et al.,in Interferons and Other Regulatory Cytokines, Wiley, New York, 1988).

INF-α and IFN-β were among the first of the cytokines to be produced byrecombinant DNA technology. For example, the amino acid and nucleotidesequence of human IFN-β [Tanaguchi et al., Gene 10(1):11-15 (1980);Houghton et al., Nucleic Acids Res. 8(13):2885-94 (1980)] made itpossible to produce recombinant human IFN-β in e.g., mammalian, insect,and yeast cells and in E. coli, that is free from viruses and othercontaminants from human sources [e.g., Ohno and Taniguchi, Nucleic AcidsRes. 10(3):967-77 (1982); Smith et al., Mol. Cell. Biol. 3(12):2156-65(1983); Demolder et al., J. Biotechnol. 32(2):179-89 (1994); Dorin etal., U.S. Pat. No. 5,814,485 (1998); Konrad et al., U.S. Pat. No.4,450,103 (1984)].

IFNs have been shown to have therapeutic value in conditions such asinflammatory, viral, and malignant diseases [e.g., see Desmyter et al.,Lancet 2(7987):645-7 (1976); Makower and Wadler, Semin. Oncol.26(6):663-71 (1999); Sturzebecher et al., J. Interferon Cytokine Res.19(11):1257-64 (1999); Zein, Cytokines Cell. Mol. Ther. 4(4):229-41(1998; Musch et al., Hepatogastroeneterology 45(24):2282-94 (1998);Wadler et al., Cancer J. Sci. Am. 4(5):331-7 (1998)]. IFN-β is amarketed drug (Betaseron, manufactured by Berlex and Avonex,manufactured by Biogen) that has been approved for use in treatment ofmultiple sclerosis (MS) [Arnason, Biomed Pharmacother 53(8):344-50,(1999); Comi et al., Mult. Scler. 1(6):317-20 (1996); Aappos, Lancet353(9171):2242-3 (1999)]. IFN-β seems to reduce the number of attackssuffered by patients with relapsing and remitting MS. Betaseron, arecombinant IFN-β expressed in E. coli, consists of 165 amino acids(missing the initial methionine) and is genetically engineered so thatit contains a serine at position 17, to replace a cysteine. It is anonglycosylated form of IFN-β. Avonex is a human IFN-β, consisting of166 amino acids that is produced by recombinant DNA techniques in CHOcells. This is a glycosylated form of IFN-β. Also, recent studies showpromising IFN efficacy in treating certain viral diseases, such asHepatitis B or C, and cancer.

Most cytokines, including IFN-β, have relatively short circulationhalf-lives since they are produced in vivo to act locally andtransiently. To use IFN-β as an effective systemic therapeutic, oneneeds relatively large doses and frequent administrations. Frequentparenteral administrations are inconvenient and painful. Further, toxicside effects are associated with IFN-β administration which are sosevere that some multiple sclerosis patents cannot tolerate thetreatment. These side effects are probably associated withadministration of a high dosage. In clinical studies it has been foundthat some patients produce antibodies to IFN-β, which neutralize itsbiological activity.

Furthermore, it has been observed that dimers and oligomers ofmicrobially produced IFN-β are formed in E. coli, rendering purificationand separation of IFN-β laborious and time consuming. It alsonecessitates several additional steps in purification and isolationprocedures such as reducing the protein during purification andreoxidizing it to restore it to its original conformation, therebyincreasing the possibility of incorrect disulfide bond formation. Inaddition, and most likely attributable to the above-listed shortcomings,microbially produced recombinant human IFN-β has also been found toexhibit consistently low specific activity. It would be desirable,therefore, to microbially produce a biologically active IFN-β proteinthat has a reduced or eliminated ability to form intermolecularcrosslinks or intramolecular bonds that cause the protein to adopt anundesirable structure.

To this end, variants of IFN-β sequences, applications and productionprocedures are known; see for example U.S. Pat. Nos. 4,450,103;4,518,584; 4,588,585; 4,737,462; 4,738,844; 4,738,845; 4,753,795;4,769,233; 4,793,995; 4,914,033; 4,959,314; 5,183,746; 5,376,567;5,545,723; 5,730,969; 5,814,485; 5,869,603 and references cited therein.

Recently, the crystal structures of recombinant murine INFβ [Senda etal., EMBO J. 11(9):3193-201 (1992); Mitsui et al., Pharmacol. Ther.58(1):93-132 (1993); Senda et al., J. Mol. Biol. 253(1):187-207 (1995);Mitsui et al., J. Interferon Cytokine Res. 17(6):319-26 (1997); all ofwhich are expressly incorporated by reference] and human INFβ [Karpusaset al., Proc. Natl. Acad. Sci. U.S.A. 94(22):11813-8 (1997); Runkel etal., Pharm. Res. 15(4):641-9 (1998); Runkel et a 273(14):8003-8 (1998);Lewerenz et al., J. Mol. Biol. 282(3):585-99 (1998); all of which areexpressly incorporated by reference] have been solved. Karpusas et al.determined the crystal structure of glycosylated human IFN-β at 2.2Angstrom resolution by molecular replacement. The molecule adopts a foldsimilar to that of the previously determined structures of murine IFN-βand human IFN-α2b, but displays several, distinct structural features.Like human IFN-α2b, INF-P contains a zinc-binding site at the interfaceof the two molecules in the asymmetric unit, however, unlike humanIFN-α2b, IFN-β dimerizes with contact surfaces from opposite sides ofthe molecule. Runkel et al. reported structural and functionaldifferences between glycosylated (IFN,β-1a) and non-glycosylated(IFN-β1b) forms of human IFN-β and suggested that the greater biologicalactivity of INF-β-1a is due to the stabilizing effect of thecarbohydrate moiety.

The available crystal structure of INFβ allows further protein designand the generation of more stable proteins or protein variants with analtered activity. Several groups have applied and experimentally testedsystematic, quantitative methods to protein design with the goal ofdeveloping general design algorithms (Hellinga et al., J. Mol. Biol.222: 763-785 (1991); Hurley et al., J. Mol. Biol. 224:1143-1154 (1992);Desjarlaisl et al., Protein Science 4:2006-2018 (1995); Harbury et al.,Proc. Natl. Acad. Sci. U.S.A. 92:8408-8412 (1995); Klemba et al., Nat.Struc. Biol. 2:368-373 (1995); Nautiyal et al., Biochemistry34:11645-11651 (1995); Betzo et al., Biochemistry 35:6955-6962 (1996);Dahiyat et al., Protein Science 5:895-903 (1996); Dahiyat et al.,Science 278:82-87 (1997); Dahiyat et al., J. Mol. Biol. 273:789-96;Dahiyat et al., Protein Sci. 6:1333-1337 (1997); Jones, Protein Science3:567-574 (1994); Konoi, et al., Proteins: Structure, Function andGenetics 19:244-255 (1994)). These algorithms consider the spatialpositioning and steric complementarity of side chains by explicitlymodeling the atoms of sequences under consideration. In particular,W098/47089, and U.S. Ser. No. 09/127,926 describe a system for proteindesign; both are expressly incorporated by reference.

A need still exists for proteins exhibiting both significant stabilityand interferon-beta activity. Accordingly, it is an object of theinvention to provide interferon-beta activity (IbA) proteins, nucleicacids and antibodies for the treatment of multiple sclerosis, cancer andviral infections.

SUMMARY OF THE INVENTION

In accordance with the objects outlined above, the present inventionprovides non-naturally occurring interferon-beta activity (IbA) proteins(e.g. the proteins are not found in nature) comprising amino acidsequences that are less than about 97% identical to human INF-β. The IbAproteins have at least one altered biological property of an INF-βprotein; for example, the IbA proteins will be more stable than IFN-βand bind to cells comprising an interferon receptor complex. Thus, theinvention provides IbA proteins with amino acid sequences that have atleast about 3-5 amino acid substitutions as compared to the INF-βsequence shown in FIG. 1 (SEQ ID NO:1).

In a further aspect, the present invention provides non-naturallyoccurring IbA conformers that have three dimensional backbone structuresthat substantially correspond to the three dimensional backbonestructure of INFβ. In one aspect, the three dimensional backbonestructure of the IbA conformer corresponds substantially to the threedimensional backbone structure of the A-chain of INFβ. In anotheraspect, the three dimensional backbone structure of the IbA conformercorresponds substantially to the three dimensional backbone structure ofthe B-chain of INF-β. The amino acid sequence of the IbA conformer andthe amino acid sequence of INF-β are less than about 97% identical. Inone aspect, at least about 90% of the non-identical amino acids are in acore region of the conformer. In other aspects, the conformer have atleast about 100% of the non-identical amino acids are in a core regionof the conformer.

In an additional aspect, the changes are selected from the amino acidresidues at positions selected from positions 6, 13, 17, 21, 56, 59, 61,62, 63, 66, 69, 84, 87, 91, 98, 102, 114, 118, 122, 129, 146, 150, 154,157, 160, and 161. In a preferred aspect, the changes are selected fromthe amino acid residues at positions selected from positions 13, 17, 56,59, 63, 66, 69, 84, 87, 91, 98, 114, 118, 122, 146, 157, and 161. In oneaspect, the changes are selected from the amino acid residues atpositions selected from positions 13, 17, 69, 84, 87, 91, 98, 118, 122,146, 157, and 161. In another aspect, the changes are selected from theamino acid residues at positions selected from positions 13, 17, 56, 84,87, 91, 114, 118, 122, and 161. Preferred embodiments include at leastabout 3-5 variations.

In a further aspect, the invention provides recombinant nucleic acidsencoding the non-naturally occurring IbA proteins, expression vectorscomprising the recombinant nucleic acids, and host cells comprising therecombinant nucleic acids and expression vectors.

In an additional aspect, the invention provides methods of producing theIbA proteins of the invention comprising culturing host cells comprisingthe recombinant nucleic acids under conditions suitable for expressionof the nucleic acids. The proteins may optionally be recovered. In afurther aspect, the invention provides pharmaceutical compositionscomprising an IbA protein of the invention and a pharmaceutical carrier.

In an additional aspect, the invention provides methods for treating anINFβ responsive condition comprising administering an IbA protein of theinvention to a patient. The INFβ condition includes multiple sclerosis,viral infection, or cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A (SEQ ID NO:1) depicts the amino acid sequence of the A-chain ofhuman INFβ as used in the determination of the crystal structure [PDBand GenBank # 1AU1; Karpusas et al., Proc. Natl. Acad. Sci. U.S.A.94(22):11813-8 (1997)] and secondary structure elements. Secondarystructure element legend: H, alpha helix (4-helix); B, residue inisolated beta bridge; E, extended strand, participates in beta ladder;G, 310 helix (3-helix); I, pi helix (5-helix); T, hydrogen bonded turn;S, bend.

FIG. 1B (SEQ ID NO:1) depicts the amino acid sequences of the B-chain ofhuman INF-β as used in the determination of the crystal structure(Karpusas et al., supra) and secondary structure elements.

FIG. 1C (SEQ ID NO:2) depicts the complete DNA sequence encoding wildtype human INF-β (GenBank accession number NM_(—)002176). The encodedsequence consists of the signaling sequence, MTNKCLLQIALLLCFSTTALS (SEQID NO:3), and the 166 amino acids that constitute the actual protein(see FIGS. 1A and 1B) (SEQ ID NO:1). The DNA sequence of 757 nucleotidesincludes this coding sequence and a non-translated region. Bases 1 to 63encode the signaling sequence; bases 64 to 561 encode the actual IFN-β;bases 562 to 564 (TGA) are stop codon; and the rest is untranslatedsequence.

FIG. 2 depicts the structure of wild type IFN-β. Presented is theA-chain from the PDB file 1AU1. The amino acid side chains indicated arethose positions included in the PDA design of CORE 1.

FIG. 3 depicts the residues for both the A-chain and B-chain of INF-βselected for PDA. The individual sets are described in detail herein.

FIG. 4A depicts the mutation pattern of IFN-β A-chain core 1 sequencesbased on the analysis of the lowest 1000 protein sequences generated byMonte Carlo analysis of A-chain IFN-β core 1 sequences (only the aminoacid residues of positions 6, 21, 55, 56, 59, 62, 63, 66, 69, 84, 87,91, 98, 122, 129, 133, 146, 150, 157, and 160 are given). All values aregiven in %. For example, at position 87, the human INF-β amino acid isleucine (see FIG. 1) (SEQ ID NO:1); in IbA proteins, 78.7% of the top1000 sequences had phenylalanine at this position, and only 18.4% of thesequences had leucine. Similarly, for position 84 (valine in human INFβ,isoleucine (40.5%) and leucine (39.4%) are preferred over valine(19.6%).

FIG. 4B (SEQ ID NO:4) depicts a preferred IbA sequence based on the PDAanalysis of IFN-β A-chain core 1 sequence. Amino acid residues differentfrom the human IFN-β (see FIG. 1) (SEQ ID NO:1) are shown in bold andare underlined.

FIG. 5A depicts the mutation pattern of IFN-β A-chain core 2 sequencesbased on the analysis of the lowest 1000 protein sequences generated byMonte Carlo analysis of A-chain IFN-β core 2 sequences (only the aminoacid residues of positions 1, 6,10, 14, 17, 21, 38, 50, 55, 56, 58, 59,61, 62, 63, 66, 69, 70, 81, 84, 87, 91, 94, 95, 98, 102, 115, 122, 125,126, 129, 130, 133, 138, 144, 146, 147, 150, 151, 153, 154, 157, 159,160, 161, 163, and 164 are given). All values are given in %. Forexample, at position 91, the human IFN-β amino acid is valine (seeFIG. 1) (SEQ ID NO:1); in IbA proteins, 81.7% of the top 1000 sequenceshad isoleucine at this position, and only 11.5% of the sequences hadvaline. Similarly, for position 98 (leucine in human IFN-β),phenylalanine (68.8%) is preferred over leucine (31.2%).

FIG. 5B (SEQ ID NO:5) depicts a preferred IbA sequence based on the PDAanalysis of IFN-β A-chain core 2 sequence. Amino acid residues differentfrom the human IFN-β (see FIG. 1) (SEQ ID NO:1) are shown in bold andare underlined.

FIG. 6A depicts the mutation pattern of IFN-β A-chain core 3 sequencesbased on the analysis of the lowest 1000 protein sequences generated byMonte Carlo analysis of A-chain IFN-β core 3 sequences (only the aminoacid residues of positions 1, 6, 10, 13, 14, 17, 18, 21, 38, 50, 55, 56,58, 59, 61, 62, 63, 66, 69, 70, 72, 74, 76, 77, 81, 84, 87, 90, 91, 94,95, 98, 102, 114, 115, 118, 122, 125, 126, 129, 130, 132, 133, 136, 138,139, 142, 143, 144, 146, 147, 150, 151, 153, 154, 157, 159, 160, 161,163, and 164 are given). All values are given in %. For example, atposition 13, the human IFN-β amino acid is serine (see FIG. 1) (SEQ IDNO: 1); in IbA proteins, 67.7% of the top 1000 sequences hadphenylalanine at this position and 31.4% of the sequences had tyrosine.None of the IbA sequences had serine at this position. Similarly, atposition 118, the human IFN-β amino acid is serine (see FIG. 1) (SEQ IDNO:1); in IbA proteins, 89.1 % of the top 1000 sequences had alanine atthis position and 10.9% of the sequences had tyrosine. None of the IbAsequences had serine at this position.

FIG. 6B (SEQ ID NO:6) depicts a preferred IbA sequence based on the PDAanalysis of IFN-β A-chain core 3 sequence. Amino acid residues differentfrom the human IFN-β (see FIG. 1) (SEQ ID NO:1) are shown in bold andare underlined.

FIG. 6C and FIG. 6D (SEQ ID NO:7-8) depict preferred IbA sequences basedon the PDA analysis of IFN-β A-chain core 3 sequence, generated not onlyby the direct MC calculation following DEE, but also those aftercleaning the MC list (C) and when running MC over the complete sequencespace starting from the ground state generated by the direct MCcalculation (D). Amino acid residues different from the human IFN-β (seeFIG. 1) (SEQ ID NO:1) are shown in bold and are underlined.

FIG. 7A depicts the mutation pattern of IFN-β A-chain core 4 sequencesbased on the analysis of the lowest 1000 protein sequences generated byMonte Carlo analysis of A-chain IFN-β core 4 sequences. See FIG. 6A fordetails of figure legend. For example, at position 17, the human IFN-βamino acid is cysteine (see FIG. 1) (SEQ ID NO:1); in IbA proteins,82.9% of the top 1000 sequences had aspartic acid at this position, 7.1%had threonine, 4.5% had alanine, 4.1% had leucine and 1.4% had valine.None of the IbA sequences had cysteine at this position.

FIG. 7B (SEQ ID NO:9) depicts a preferred IbA sequence based on the PDAanalysis of IFN-β A-chain core 4 sequence. Amino acid residues differentfrom the human IFN-β (see FIG. 1) (SEQ ID NO:1) are shown in bold andare underlined.

FIG. 7C and FIG. 7D (SEQ ID NOS:10-11) depict preferred IbA sequencesbased on the PDA analysis of IFN-β A-chain core 4 sequence, generatednot only by the direct MC calculation following DEE, but also thoseafter cleaning the MC list (C) and when running MC over the completesequence space starting from the ground state generated by the direct MCcalculation (D). Amino acid residues different from the human IFN-β (seeFIG. 1) (SEQ ID NO:1) are shown in bold and are underlined.

FIG. 8A depicts the mutation pattern of IFN-β A-chain core 5 sequencesbased on the analysis of the lowest 1000 protein sequences generated byMonte Carlo analysis of A-chain IFN-β core 5 sequences. See FIG. 6A fordetails of figure legend. For example, at position 84, the human IFN-βamino acid is valine (see FIG. 1) (SEQ ID NO:1); in IbA proteins, 99.5%of the top 1000 sequences had isoleucine at this position and 0.5% hadleucine. None of the IbA sequences had valine at this position.

FIG. 8B (SEQ ID NO:12) depicts a preferred IbA sequence based on the PDAanalysis of IFN-β A-chain core 5 sequence. Amino acid residues differentfrom the human IFN-p (see FIG. 1) (SEQ ID NO:1) are shown in bold andare underlined.

FIG. 8C and FIG. 8D (SEQ ID NOS:13-14) depict preferred IbA sequencesbased on the PDA analysis of IFN-β A-chain core 5 sequence, generatednot only by the direct MC calculation following DEE, but also thoseafter cleaning the MC list (C) and when running MC over the completesequence space starting from the ground state generated by the direct MCcalculation (D). Amino acid residues different from the human IFN-β (seeFIG. 1) (SEQ ID NO:1) are shown in bold and are underlined.

FIG. 9A depicts the mutation pattern of IFN-β A-chain core 6 sequencesbased on the analysis of the lowest 1000 protein sequences generated byMonte Carlo analysis of A-chain IFN-β core 6 sequences. See FIG. 6A fordetails of figure legend. For example, at position 118, the human IFN-βamino acid is serine (see FIG. 1) (SEQ ID NO:1); in IbA proteins, 100%of the top 1000 sequences had alanine. None of the IbA sequences hadserine at this position.

FIG. 9B (SEQ ID NO:15) depicts a preferred IbA sequence based on the PDAanalysis of IFN-β A-chain core 6 sequence. Amino acid residues differentfrom the human IFN-β (see FIG. 1) (SEQ ID NO:1) are shown in bold andare underlined.

FIG. 9C and FIG. 9D (SEQ ID NOS:16-17) depict preferred IbA sequencesbased on the PDA analysis of IFN-β A-chain core 6 sequence, generatednot only by the direct MC calculation following DEE, but also thoseafter cleaning the MC list (C) and when running MC over the completesequence space starting from the ground state generated by the direct MCcalculation (D). Amino acid residues different from the human IFN-β (seeFIG. 1) (SEQ ID NO:1) are shown in bold and are underlined.

FIG. 10A depicts the mutation pattern of IFN-β B-chain core 1 sequencesbased on the analysis of the lowest 1000 protein sequences generated byMonte Carlo analysis of B-chain IFN-β core 1 sequences (only the aminoacid residues of positions 6, 21, 55, 56, 59, 62, 63, 66, 69, 84, 87,91, 98, 122, 129, 133, 146, 150, 157, and 160 are given). All values aregiven in %. For example, at position 87, the human IFN-β amino acid isleucine (see FIG. 1) (SEQ ID NO:1); in IbA proteins, 74.6% of the top1000 sequences had phenylalanine at this position, and only 21.5% of thesequences had leucine. Similarly, for position 84 (valine in humanIFN-β), isoleucine (62.3%) is preferred over valine (25.4%).

FIG. 10B (SEQ ID NO:18) depicts a preferred IbA sequence based on thePDA analysis of IFN-β B-chain core 1 sequence. Amino acid residuesdifferent from the human IFN-β (see FIG. 1) SEQ ID NO:l) are shown inbold and are underlined.

FIG. 11A depicts the mutation pattern of IFN-β B-chain core 2 sequencesbased on the analysis of the lowest 1000 protein sequences generated byMonte Carlo analysis of B-chain IFN-β core 2 sequences (only the aminoacid residues of positions 1, 6, 10, 14, 17, 21, 38, 50, 55, 56, 58, 59,61, 62, 63, 66, 69, 70, 81, 84, 87, 91, 94, 95, 98, 102, 115, 122, 125,126, 129, 130, 133, 138, 144, 146, 147, 150, 151, 153, 154, 157, 159,160, 161, 163, and 164 are given). All values are given in %. Forexample, at position 56, the human IFN-β amino acid is alanine (seeFIG. 1) (SEQ ID NO:1); in IbA proteins, 97.6% of the top 1000 sequenceshad leucine at this position, and only 2.4% of the sequences hadalanine. Similarly, for position 91 (valine in human IFN-β), isoleucine(68.5%) and leucine (27.7%) are preferred over valine (3.8%).

FIG. 11B (SEQ ID NO:19) depicts a preferred IbA sequence based on thePDA analysis of IFN-β B-chain core 2 sequence. Amino acid residuesdifferent from the human IFN-β (see FIG. 1) (SEQ ID NO:1) are shown inbold and are underlined.

FIG. 12A depicts the mutation pattern of IFN-β B-chain core 3 sequencesbased on the analysis of the lowest 1000 protein sequences generated byMonte Carlo analysis of B-chain IFN-β core 3 sequences (only the aminoacid residues of positions 1, 6, 10, 13, 14, 15, 17, 21, 38, 50, 55, 56,58, 10 59, 61, 62, 63, 66, 69, 70, 72, 74, 76, 77, 81, 84, 87, 90, 91,94, 95, 98, 102, 114, 115, 118, 122, 125, 126, 129, 130, 132, 133, 136,138, 139, 142, 143, 144, 146, 147, 150, 151, 153, 154, 157, 159, 160,161, 163, and 164 are given). All values are given in %. For example, atposition 13, the human IFN-β amino acid is serine (see FIG. 1); in IbAproteins, 92.7% of the top 1000 sequences had leucine at this positionand 7.3% of the sequences had alanine. None of the IbA sequences hadserine at this position. Similarly, at position 118, the human IFN-βamino acid is serine (see FIG. 1) (SEQ ID NO:1); in IbA proteins, 100%of the top 1000 sequences had leucine at this position.

FIG. 12B (SEQ ID NO:20) depicts a preferred IbA sequence based on thePDA analysis of IFN-β B-chain core 3 sequence. Amino acid residuesdifferent from the human IFN-β (see FIG. 1) (SEQ ID NO:1) are shown inbold and are underlined.

FIG. 13A depicts the mutation pattern of IFN-β B-chain core 4 sequencesbased on the analysis of the lowest 1000 protein sequences generated byMonte Carlo analysis of B-chain IFN-β core 4 sequences. See FIG. 12A fordetails of figure legend. For example, at position 56, the human IFN-βamino acid is alanine (see FIG. 1) (SEQ ID NO:l); in IbA proteins, 97.7%of the top 1000 sequences had leucine at this position and only 2.3% hadalanine. Similarly, at position 114, the human IFN-β amino acid isglycine (see FIG. 1) (SEQ ID NO:1); in IbA proteins, 100% of the top1000 sequences had phenylalanine at this position.

FIG. 13B (SEQ ID NO:21) depicts a preferred IbA sequence based on thePDA analysis of IFN-β B-chain core 4 sequence. Amino acid residuesdifferent from the human IFN-β (see FIG. 1) (SEQ ID NO:1) are shown inbold and are underlined.

FIG. 14A depicts the mutation pattern of IFN-β B-chain core 5 sequencesbased on the analysis of the lowest 1000 protein sequences generated byMonte Carlo analysis of B-chain IFN-β core 5 sequences (only the aminoacid residues of positions 1, 6, 10, 13, 14, 17, 18, 21, 38, 50, 55, 56,58, 59, 61, 62, 63, 66, 69, 70, 72, 74, 76, 77, 81, 84, 87, 90, 91, 94,95, 98, 102, 114, 115, 118, 122, 125, 126, 129, 130, 132, 133, 136, 138,139, 142, 143, 144, 146, 147, 150, 151, 153, 154, 157, 159, 160, 161,163, and 164 are given). For example, at position 56, the human IFN-βamino acid is alanine (see FIG. 1) (SEQ ID NO:1); in IbA proteins, 97.6%of the top 1000 sequences had leucine at this position and only 2.4% hadalanine. Similarly, at position 114, the human IFN-β amino acid isglycine (see FIG. 1) (SEQ ID NO:1); in IbA proteins, 100% of the top1000 sequences had leucine at this position.

FIG. 14B (SEQ ID NO:1) depicts a preferred IbA sequence based on the PDAanalysis of IFN-β B-chain core 5 sequence. Amino acid residues differentfrom the human IFN-β (see FIG. 1) (SEQ ID NO:1) are shown in bold andare underlined.

FIG. 15A depicts the mutation pattern of IFN-β B-chain core 6 sequencesbased on the analysis of the lowest 1000 protein sequences generated byMonte Carlo analysis of B-chain IFN-β core 6 sequences. See FIG. 14A fordetails of figure legend. For example, at position 118, the human IFN-βamino acid is serine (see FIG. 1) (SEQ ID NO:1); in IbA proteins, 99.4%of the top 1000 sequences had glutamic acid at this position and 0.6%had alanine. None of the IbA sequences had serine at this position.Similarly, for position 161 (threonine in human IFN-β), glutamic acid(86.4%) is preferred over threonine (12.1 %).

FIG. 15B (SEQ ID NO:23) depicts a preferred IbA sequence based on thePDA analysis of IFN-β B-chain core 6 sequence. Amino acid residuesdifferent from the human IFN-β (see FIG. 1) (SEQ ID NO:1) are shown inbold and are underlined.

FIG. 16A depicts the mutation pattern of IFN-β, B-chain core 7 sequencesbased on the analysis of the lowest 1000 protein sequences generated byMonte Carlo analysis of B-chain IFN-β core 7 sequences. See FIG. 14A fordetails of figure legend. For example, at position 17, the human IFN-βamino acid is cysteine (see FIG. 1) (SEQ ID NO:1); in IbA proteins,32.8% of the top 1000 sequences had threonine at this position, 31 % hadalanine, 29% had aspartic acid, 5% had glutamic acid, 1.4% had serine,and 0.8% had glycine. None of the IbA sequences had cysteine at thisposition.

FIG. 16B (SEQ ID NO:24) depicts a preferred IbA sequence based on thePDA analysis of IFN-β B-chain core 7 sequence. Amino acid residuesdifferent from the human IFN-β (see FIG. 1) (SEQ ID NO:1) are shown inbold and are underlined.

FIG. 17 depicts the synthesis of a full-length gene and all possiblemutations by PCR. Overlapping oligonucleotides corresponding to thefull-length gene (black bar, Step 1) and comprising one or more desiredmutations are synthesized, heated and annealed. Addition of DNApolymerase to the annealed oligonucleotdes results in the 5′ to 3′synthesis of DNA (Step 2) to produce longer DNA fragments (Step 3).Repeated cycles of heating, annealing, and DNA synthesis (Step 4) resultin the production of longer DNA, including some full-length molecules.These can be selected by a second round of PCR using primers (indicatedby arrows) corresponding to the end of the full-length gene (Step 5).

FIG. 18 depicts a preferred scheme for synthesizing an IbA library ofthe invention. The wild type gene, or any starting gene, such as thegene for the global minima gene, can be used. Oligonucleotidescomprising sequences that encode different amino acids at the differentvariant positions (indicated in the Figure by box 1, box 2, and box 3)can be used during PCR. Those primers can be used in combination withstandard primers. This generally requires fewer oligonucleotides and canresult in fewer errors.

FIG. 19 depicts an overlapping extension method. At the top of FIG. 19is the template DNA showing the locations of the regions to be mutated(black boxes) and the binding sites of the relevant primers (arrows).The primers R1 and R2 represent a pool of primers, each containing adifferent mutation; as described herein, this may be done usingdifferent ratios of primers if desired. The variant position is flankedby regions of homology sufficient to get hybridization. Thus, as shownin this example, oligos R1 and F2 comprise a region of homology and sodo oligos R2 and F3. In this example, three separate PCR reactions aredone for step 1. The first reaction contains the template plus oligos F1and R1. The second reaction contains template plus oligos F2 and R2, andthe third contains the template and oligos F3 and R3. The reactionproducts are shown. In Step 2, the products from Step 1 tube 1 and Step1 tube 2 are taken. After purification away from the primers, these areadded to a fresh PCR reaction together with F1 and R4. During thedenaturation phase of the PCR, the overlapping regions anneal and thesecond strand is synthesized. The product is then amplified by theoutside primers, F1 and R4. In Step 3, the purified product from Step 2is used in a third PCR reaction, together with the product of Step 1,tube 3 and the primers F1 and R3. The final product corresponds to thefull length gene and contains the required mutations. Alternatively,Step 2 and Step 3 can be performed in one PCR reaction.

FIG. 20 depicts a ligation of PCR reaction products to synthesize thelibraries of the invention. In this technique, the primers also containan endonuclease restriction site (RE), either generating blunt ends, 5′overhanging ends or 3′ overhanging ends. We set up three separate PCRreactions for Step 1. The first reaction contains the template plusoligos F1 and R1. The second reaction contains template plus oligos F2and R2, and the third contains the template and oligos F3 and R3. Thereaction products are shown. In Step 2, the products of Step 1 arepurified and then digested with the appropriate restrictionendonuclease. The digestion products from Step 2, tube 1 and Step 2,tube 2 are ligated together with DNA ligase (Step 3). The products arethen amplified in Step 4 using oligos F1 and R4. The whole process isthen repeated by digesting the amplified products, ligating them to thedigested products of Step 2, tube 3, and then amplifying the finalproduct using oligos F1 and R3. It would also be possible to ligate allthree PCR products from Step 1 together in one reaction, providing thetwo restriction sites (RE1 and RE2) were different.

FIG. 21 depicts blunt end ligation of PCR products. In this technique,oligos such as F2 and R1 or R2 and F3 do not overlap, but they abut.Again three separate PCR reactions are performed. The products from tube1 and tube 2 (see FIG. 20, Step 1) are ligated, and then amplified withoutside primers F1 and R4. This product is then ligated with the productfrom Step 1, tube 3. The final products are then amplified with primersF1 and R3.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to novel proteins and nucleic acidspossessing interferon-beta activity (sometimes referred to herein as“IbA proteins” and “IbA nucleic acids”). The proteins are generatedusing a system previously described in WO98/47089 and U.S. Ser. Nos.09/058,459, 09/127,926, 60/104,612, 60/158,700, 09/419,351, 60/181,630,60/186,904, and U.S patent application, entitled Protein DesignAutomation For Protein Libraries (Filed: Apr. 14, 2000; Inventor: BassilDahiyat), all of which are expressly incorporated by reference in theirentirety, that is a computational modeling system that allows thegeneration of extremely stable proteins without necessarily disturbingthe biological functions of the protein itself. In this way, novel IbAproteins and nucleic acids are generated, that can have a plurality ofmutations in comparison to the wild-type enzyme yet retain significantactivity.

Generally, there are a variety of computational methods that can be usedto generate the IbA proteins of the invention. In a preferredembodiment, sequence based methods are used. Alternatively, structurebased methods, such as PDA, described in detail below, are used.

Similarly, molecular dynamics calculations can be used tocomputationally screen sequences by individually calculating mutantsequence scores and compiling a rank ordered list.

In a preferred embodiment, residue pair potentials can be used to scoresequences (Miyazawa et al., Macromolecules 18(3):534-552 (1985),expressly incorporated by reference) during computational screening.

In a preferred embodiment, sequence profile scores (Bowie et al.,Science 253(5016):164-70 (1991), incorporated by reference) and/orpotentials of mean force (Hendlich et al., J. Mol. Biol. 216(1):167-180(1990), also incorporated by reference) can also be calculated to scoresequences. These methods assess the match between a sequence and a 3Dprotein structure and hence can act to screen for fidelity to theprotein structure. By using different scoring functions to ranksequences, different regions of sequence space can be sampled in thecomputational screen.

Furthermore, scoring functions can be used to screen for sequences thatwould create metal or co-factor binding sites in the protein (Hellinga,Fold Des. 3(1):R1-8 (1998), hereby expressly incorporated by reference).Similarly, scoring functions can be used to screen for sequences thatwould create disulfide bonds in the protein. These potentials attempt tospecifically modify a protein structure to introduce a new structuralmotif.

In a preferred embodiment, sequence and/or structural alignment programscan be used to generate the IbA proteins of the invention. As is knownin the art, there are a number of sequence-based alignment programs;including for example, Smith-Waterman searches, Needleman-Wunsch, DoubleAffine Smith-Waterman, frame search, Gribskov/GCG profile search,Gribskov/GCG profile scan, profile frame search, Bucher generalizedprofiles, Hidden Markov models, Hframe, Double Frame, Blast, Psi-Blast,Clustal, and GeneWise.

As is known in the art, there are a number of sequence alignmentmethodologies that can be used. For example, sequence homology basedalignment methods can be used to create sequence alignments of proteinsrelated to the target structure (Altschul et al., J. Mol. Biol.215(3):403-410 (1990), Altschul et al., Nucleic Acids Res. 25:3389-3402(1997), both incorporated by reference). These sequence alignments arethen examined to determine the observed sequence variations. Thesesequence variations are tabulated to define a set of IbA proteins.

Sequence based alignments can be used in a variety of ways. For example,a number of related proteins can be aligned, as is known in the art, andthe “variable” and “conserved” residues defined; that is, the residuesthat vary or remain identical between the family members can be defined.These results can be used to generate a probability table, as outlinedbelow. Similarly, these sequence variations can be tabulated and asecondary library defined from them as defined below. Alternatively, theallowed sequence variations can be used to define the amino acidsconsidered at each position during the computational screening. Anothervariation is to bias the score for amino acids that occur in thesequence alignment, thereby increasing the likelihood that they arefound during computational screening but still allowing consideration ofother amino acids. This bias would result in a focused library of IbAproteins but would not eliminate from consideration amino acids notfound in the alignment. In addition, a number of other types of bias maybe introduced. For example, diversity may be forced; that is, a“conserved” residue is chosen and altered to force diversity on theprotein and thus sample a greater portion of the sequence space.Alternatively, the positions of high variability between family members(i.e. low conservation) can be randomized, either using all or a subsetof amino acids. Similarly, outlier residues, either positional outliersor side chain outliers, may be eliminated.

Similarly, structural alignment of structurally related proteins can bedone to generate sequence alignments (Orengo et al., Structure5(8):1093-108 (1997); Holm et al., Nucleic Acids Res. 26(1):316-9(1998), both of which are incorporated by reference). These sequencealignments can then be examined to determine the observed sequencevariations. Libraries can be generated by predicting secondary structurefrom sequence, and then selecting sequences that are compatible with thepredicted secondary structure. There are a number of secondary structureprediction methods such as helix-coil transition theory (Munoz andSerrano, Biopolymers 41:495, 1997), neural networks, local structurealignment and others (e.g., see in Selbig et al., Bioinformatics15:1039-46, 1999).

Similarly, as outlined above, other computational methods are known,including, but not limited to, sequence profiling [Bowie and Eisenberg,Science 253(5016):164-70, (1991)], rotamer library selections [Dahiyatand Mayo, Protein Sci. 5(5):895-903 (1996); Dahiyat and Mayo, Science278(5335):82-7 (1997); Desjarlais and Handel, Protein Science4:2006-2018 (1995); Harbury et Proc. Natl. Acad. Sci. U.S.A.92(18):8408-8412 (1995); Kono et al., Proteins: Structure, Function andGenetics 19:244-255 (1994); Hellinga and Richards, Proc. Natl. Acad.Sci. U.S.A. 91:5803-5807 (1994)]; and residue pair potentials [Jones,Protein Science 3: 567-574, (1994)]; PROSA [Heindlich et al., J. Mol.Biol. 216:167-180 (1990)]; THREADER [Jones et al., Nature 358:86-89(1992)], and other inverse folding methods such as those described bySimons et al. [Proteins, 34:535-543, (1999)], Levitt and Gerstein [Proc.Natl. Acad. Sci. U.S.A., 95:5913-5920, (1998)], Godzik and Skolnick[Proc. Natl. Acad. Sci. U.S.A., 89:12098-102, (1992)], Godzik et al. [J.Mol. Biol. 227:227-38, (1992)], and other profile methods [Gribskov etal. Proc. Natl. Acad. Sci. U.S.A. 84:4355-4358 (1987) and Fischer andEisenberg, Protein Sci. 5:947-955 (1996), Rice and Eisenberg J. Mol.Biol. 267:1026-1038(1997)], all of which are expressly incorporated byreference. In addition, other computational methods such as thosedescribed by Koehl and Levitt (J. Mol. Biol. 293:1161-1181 (1999); J.Mol. Biol. 293:1183-1193 (1999); expressly incorporated by reference)can be used to create a protein sequence library which can optionallythen be used to generate a smaller secondary library for use inexperimental screening for improved properties and function. Inaddition, there are computational methods based on forcefieldcalculations such as SCMF that can be used as well for SCMF, see Delarueet al. Pac. Symp. Biocomput. 109-21 (1997); Koehl et al., J. Mol. Biol.239:249-75 (1994); Koehl et al., Nat. Struct. Biol. 2:163-70 (1995);Koehl et al., Curr. Opin. Struct. Biol. 6:222-6 (1996); Koehl et al., J.Biol. 293:1183-93 (1999); Koehl et al., J. Mol. Biol. 293:1161-81(1999); Lee J., Mol. Biol.236:918-39 (1994); and Vasquez Biopolymers36:53-70 (1995); all of which are expressly incorporated by reference.Other forcefield calculations that can be used to optimize theconformation of a sequence within a computational method, or to generatede novo optimized sequences as outlined herein include, but are notlimited to, OPLS-AA [Jorgensen et al., J. Am. Chem. Soc. 118:11225-11236(1996); Jorgensen, W. L.; BOSS, Version 4.1; Yale University: New Haven,CT (1999)]; OPLS [Jorgensen et al., J. Am. Chem. Soc.110:1657ff (1988);Jorgensen et al., J. Am. Chem. Soc.112:4768ff (1990)]; UNRES (UnitedResidue Forcefield; Liwo et al., Protein Science 2:1697-1714 (1993);Liwo et al., Protein Science 2:1715-1731 (1993); Liwo et al., J. Comp.Chem. 18:849-873 (1997); Liwo et al. Comp. Chem. 18:874-884 (1997); Liwoet al., J. Comp. Chem. 19:259-276 (1998); Forcefield for ProteinStructure Prediction (Liwo et al., Proc. Natl. Acad. Sci. U.S.A.96:5482-5485 (1999)]; ECEPP/3 [Liwo et al., J Protein Chem. 13(4):375-80(1994)]; AMBER 1.1 force field (Weiner et a Am. Chem. Soc. 106:765-784);AMBER 3.0 force field [U. C. Singh et al., Proc. Natl. Acad. Sci. U.S.A.82:755-759 (1985)]; CHARMM and CHARMM22 (Brooks et al., J. Comp. Chem.4:187-217); cvff3.0 [Dauber-Osguthorpe et al., Proteins: Structure,Function and Genetics, 4:31-47 (1988)]; cff99:1 (Maple et al., J. Comp.Chem. 15:162-182); also, the DISCOVER (cvff and cff91) and AMBERforcefields are used in the INSIGHT molecular modeling package(Biosym/MSI, San Diego Calif.) and HARMM is used in the QUANTA molecularmodeling package (Biosym/MSI, San Diego Calif.), all of which areexpressly incorporated by reference. In fact, as is outlined below,these forcefield methods may be used to generate the secondary librarydirectly; that is, no primary library is generated; rather, thesemethods can be used to generate a probability table from which thesecondary library is directly generated.

In a preferred embodiment, the computational method used to generate theprimary library is Protein Design Automation (PDA), as is described inU.S. Ser. Nos. 60/061,097, 60/043,464, 60/054,678, 09/127,926,60/104,612, 60/158,700, 09/419,351, 60/181,630, 60/186,904, U.S patentapplication, entitled Protein Design Automation For Protein Libraries(Filed: Apr. 14, 2000; Inventor: Bassil Dahiyat) and PCT US98/07254, allof which are expressly incorporated herein by reference. Briefly, PDAcan be described as follows. A known protein structure is used as thestarting point. The residues to be optimized are then identified, whichmay be the entire sequence or subset(s) thereof. The side chains of anypositions to be varied are then removed. The resulting structureconsisting of the protein backbone and the remaining sidechains iscalled the template. Each variable residue position is then preferablyclassified as a core residue, a surface residue, or a boundary residue;each classification defines a subset of possible amino acid residues forthe position (for example, core residues generally will be selected fromthe set of hydrophobic residues, surface residues generally will beselected from the hydrophilic residues, and boundary residues may beeither). Each amino acid can be represented by a discrete set of allallowed conformers of each side chain, called rotamers. Thus, to arriveat an optimal sequence for a backbone, all possible sequences ofrotamers must be screened, where each backbone position can be occupiedeither by each amino acid in all its possible rotameric states, or asubset of amino acids, and thus a subset of rotamers.

Two sets of interactions are then calculated for each rotamer at everyposition: the interaction of the rotamer side chain with all or part ofthe backbone (the “singles” energy, also called the rotamer/template orrotamer/backbone energy), and the interaction of the rotamer side chainwith all other possible rotamers at every other position or a subset ofthe other positions (the “doubles” energy, also called therotamer/rotamer energy). The energy of each of these interactions iscalculated through the use of a variety of scoring functions, whichinclude the energy of van der Waal's forces, the energy of hydrogenbonding, the energy of secondary structure propensity, the energy ofsurface area solvation and the electrostatics. Thus, the total energy ofeach rotamer interaction, both with the backbone and other rotamers, iscalculated, and stored in a matrix form.

The discrete nature of rotamer sets allows a simple calculation of thenumber of rotamer sequences to be tested. A backbone of length n with mpossible rotamers per position will have m^(n) possible rotamersequences, a number which grows exponentially with sequence length andrenders the calculations either unwieldy or impossible in real time.Accordingly, to solve this combinatorial search problem, a “Dead EndElimination” (DEE) calculation is performed. The DEE calculation isbased on the fact that if the worst total interaction of a first rotameris still better than the best total interaction of a second rotamer,then the second rotamer cannot be part of the global optimum solution.Since the energies of all rotamers have already been calculated, the DEEapproach only requires sums over the sequence length to test andeliminate rotamers, which speeds up the calculations considerably. DEEcan be rerun comparing pairs of rotamers, or combinations of rotamers,which will eventually result in the determination of a single sequencewhich represents the global optimum energy.

Once the global solution has been found, a Monte Carlo search may bedone to generate a rank-ordered list of sequences in the neighborhood ofthe DEE solution. Starting at the DEE solution, random positions arechanged to other rotamers, and the new sequence energy is calculated. Ifthe new sequence meets the criteria for acceptance, it is used as astarting point for another jump. After a predetermined number of jumps,a rank-ordered list of sequences is generated. Monte Carlo searching isa sampling technique to explore sequence space around the global minimumor to find new local minima distant in sequence space. As is moreadditionally outlined below, there are other sampling techniques thatcan be used, including Boltzman sampling, genetic algorithm techniquesand simulated annealing. In addition, for all the sampling techniques,the kinds of jumps allowed can be altered (e.g. random jumps to randomresidues, biased jumps (to or away from wild-type, for example), jumpsto biased residues (to or away from similar residues, for example),etc.). Similarly, for all the sampling techniques, the acceptancecriteria of whether a sampling jump is accepted can be altered.

As outlined in U.S. Ser. No. 09/127,926, the protein backbone(comprising (for a naturally occuring protein) the nitrogen, thecarbonyl carbon, the α-carbon, and the carbonyl oxygen, along with thedirection of the vector from the α-carbon to the β-carbon) may bealtered prior to the computational analysis, by varying a set ofparameters called supersecondary structure parameters.

Once a protein structure backbone is generated (with alterations, asoutlined above) and input into the computer, explicit hydrogens areadded if not included within the structure (for example, if thestructure was generated by X-ray crystallography, hydrogens must beadded). After hydrogen addition, energy minimization of the structure isrun, to relax the hydrogens as well as the other atoms, bond angles andbond lengths. In a preferred embodiment, this is done by doing a numberof steps of conjugate gradient minimization [Mayo et al., J. Phys. Chem.94:8897 (1990)] of atomic coordinate positions to minimize the Dreidingforce field with no electrostatics. Generally from about 10 to about 250steps is preferred, with about 50 being most preferred.

The protein backbone structure contains at least one variable residueposition. As is known in the art, the residues, or amino acids, ofproteins are generally sequentially numbered starting with theN-terminus of the protein. Thus a protein having a methionine at it'sN-terminus is said to have a methionine at residue or amino acidposition 1, with the next residues as 2, 3, 4, etc. At each position,the wild type (i.e. naturally occuring) protein may have one of at least20 amino acids, in any number of rotamers. By “variable residueposition” herein is meant an amino acid position of the protein to bedesigned that is not fixed in the design method as a specific residue orrotamer, generally the wild-type residue or rotamer.

In a preferred embodiment, all of the residue positions of the proteinare variable. That is, every amino acid side chain may be altered in themethods of the present invention. This is particularly desirable forsmaller proteins, although the present methods allow the design oflarger proteins as well. While there is no theoretical limit to thelength of the protein which may be designed this way, there is apractical computational limit.

In an alternate preferred embodiment, only some of the residue positionsof the protein are variable, and the remainder are “fixed”, that is,they are identified in the three dimensional structure as being in a setconformation. In some embodiments, a fixed position is left in itsoriginal conformation (which may or may not correlate to a specificrotamer of the rotamer library being used). Alternatively, residues maybe fixed as a non-wild type residue; for example, when knownsite-directed mutagenesis techniques have shown that a particularresidue is desirable (for example, to eliminate a proteolytic site oralter the substrate specificity of an enzyme), the residue may be fixedas a particular amino acid. Alternatively, the methods of the presentinvention may be used to evaluate mutations de novo, as is discussedbelow. In an alternate preferred embodiment, a fixed position may be“floated”; the amino acid at that position is fixed, but differentrotamers of that amino acid are tested. In this embodiment, the variableresidues may be at least one, or anywhere from 0.1% to 99.9% of thetotal number of residues. Thus, for example, it may be possible tochange only a few (or one) residues, or most of the residues, with allpossibilities in between.

In a preferred embodiment, residues which can be fixed include, but arenot limited to, structurally or biologically functional residues;alternatively, biologically functional residues may specifically not befixed. For example, residues which are known to be important forbiological activity, such as the residues which the binding site for abinding partner (ligand/receptor, antigen/antibody, etc.),phosphorylation or glycosylation sites which are crucial to biologicalfunction, or structurally important residues, such as disulfide bridges,metal binding sites, critical hydrogen bonding residues, residuescritical for backbone conformation such as proline or glycine, residuescritical for packing interactions, etc. may all be fixed in their aminoacid identity and a single rotamer conformation, or “floated”, whichonly fixes the identity but not the rotamer conformation.

Similarly, residues which may be chosen as variable residues may bethose that confer undesirable biological attributes, such assusceptibility to proteolytic degradation, dimerization or aggregationsites, glycosylabon sites which may lead to immune responses, unwantedbinding activity, unwanted allostery, undesirable enzyme activity butwith a preservation of binding, etc.

In a preferred embodiment, each variable position is classified aseither a core, surface or boundary residue position, although in somecases, as explained below, the variable position may be set to glycineto minimize backbone strain. In addition, as outlined herein, residuesneed not be classified, they can be chosen as variable and any set ofamino acids may be used. Any combination of core, surface and boundarypositions can be utilized: core, surface and boundary residues; core andsurface residues; core and boundary residues, and surface and boundaryresidues, as well as core residues alone, surface residues alone, orboundary residues alone.

The classification of residue positions as core, surface or boundary maybe done in several ways, as will be appreciated by those in the art. Ina preferred embodiment, the classification is done via a visual scan ofthe original protein backbone structure, including the side chains, andassigning a classification based on a subjective evaluation of oneskilled in the art of protein modelling. Alternatively, a preferredembodiment utilizes an assessment of the orientation of the Cα-Cβvectors relative to a solvent accessible surface computed using only thetemplate Cα atoms, as outlined in U.S. Ser. Nos. 60/061,097, 60/043,464,60/054,678, 09/127,926 60/104,612, 60/158,700, 09/419,351, 60/181,630,60/186,904, U.S patent application, entitled Protein Design AutomationFor Protein Libraries (Filed: Apr. 14, 2000; Inventor: Bassil Dahiyat)and PCT US98/07254. Alternatively, a surface area calculation can bedone.

Suitable core and boundary positions for IbA proteins are outlinedbelow.

Once each variable position is classified as either core, surface orboundary, a set of amino acid side chains, and thus a set of rotamers,is assigned to each position. That is, the set of possible amino acidside chains that the program will allow to be considered at anyparticular position is chosen. Subsequently, once the possible aminoacid side chains are chosen, the set of rotamers that will be evaluatedat a particular position can be determined. Thus, a core residue willgenerally be selected from the group of hydrophobic residues consistingof alanine, valine, isoleucine, leucine, phenylalanine, tyrosine,tryptophan, and methionine (in some embodiments, when the a scalingfactor of the van der Waals scoring function, described below, is low,methionine is removed from the set), and the rotamer set for each coreposition potentially includes rotamers for these eight amino acid sidechains (all the rotamers if a backbone independent library is used, andsubsets if a rotamer dependent backbone is used). Similarly, surfacepositions are generally selected from the group of hydrophilic residuesconsisting of alanine, serine, threonine, aspartic acid, asparagine,glutamine, glutamic acid, arginine, lysine and histidine. The rotamerset for each surface position thus includes rotamers for these tenresidues. Finally, boundary positions are generally chosen from alanine,serine, threonine, aspartic acid, asparagine, glutamine, glutamic acid,arginine, lysine histidine, valine, isoleucine, leucine, phenylalanine,tyrosine, tryptophan, and methionine. The rotamer set for each boundaryposition thus potentially includes every rotamer for these seventeenresidues (assuming cysteine, glycine and proline are not used, althoughthey can be). Additionally, in some preferred embodiments, a set of 18naturally occuring amino acids (all except cysteine and proline, whichare known to be particularly disruptive) are used.

Thus, as will be appreciated by those in the art, there is acomputational benefit to classifying the residue positions, as itdecreases the number of calculations. It should also be noted that theremay be situations where the sets of core, boundary and surface residuesare altered from those described above; for example, under somecircumstances, one or more amino acids is either added or subtractedfrom the set of allowed amino acids. For example, some proteins whichdimerize or multmerize, or have ligand binding sites, may containhydrophobic surface residues, etc. In addition, residues that do notallow helix “capping” or the favorable interaction with an a-helixdipole may be subtracted from a set of allowed residues. Thismodification of amino acid groups is done on a residue by residue basis.

In a preferred embodiment, proline, cysteine and glycine are notincluded in the list of possible amino acid side chains, and thus therotamers for these side chains are not used. However, in a preferredembodiment, when the variable residue position has a φ angle (that is,the dihedral angle defined by 1) the carbonyl carbon of the precedingamino acid; 2) the nitrogen atom of the current residue; 3) the α-carbonof the current residue; and 4) the carbonyl carbon of the currentresidue) greater than 0°, the position is set to glycine to minimizebackbone strain.

Once the group of potential rotamers is assigned for each variableresidue position, processing proceeds as outlined in U.S. Ser. No.09/127, 926 and PCT US98/07254. This processing step entails analyzinginteractions of the rotamers with each other and with the proteinbackbone to generate optimized protein sequences. Simplistically, theprocessing initially comprises the use of a number of scoring functionsto calculate energies of interactions of the rotamers, either to thebackbone itself or other rotamers. Preferred PDA scoring functionsinclude, but are not limited to, a Van der Waals potential scoringfunction, a hydrogen bond potential scoring function, an atomicsolvation scoring function, a secondary structure propensity scoringfunction and an electrostatic scoring function. As is further describedbelow, at least one scoring function is used to score each position,although the scoring functions may differ depending on the positionclassification or other considerations, like favorable interaction withan α-helix dipole. As outlined below, the total energy which is used inthe calculations is the sum of the energy of each scoring function usedat a particular position, as is generally shown in Equation 1:

E _(total) =nE _(vdw) +nE _(as) +nE _(h-bonding) +nE _(ss) +nE_(elec)  Equation 1

In Equation 1, the total energy is the sum of the energy of the van derWaals potential (E_(vdw)), the energy of atomic solvation (E_(as)), theenergy of hydrogen bonding (E_(h-bonding)), the energy of secondarystructure (E_(ss)) and the energy of electrostatic interaction(E_(elec)). The term n is either 0 or 1, depending on whether the termis to be considered for the particular residue position.

As outlined in U.S. Ser. Nos. 60/061,097, 60/043,464, 60/054,678,09/127,926, 60/104,612, 60/158,700, 09/419,351, 60/181,630, 60/186,904,U.S patent application, entitled Protein Design Automation For ProteinLibraries (Filed: Apr. 14, 2000; Inventor: Bassil Dahiyat) and PCTUS98/07254, any combination of these scoring functions, either alone orin combination, may be used. Once the scoring functions to be used areidentified for each variable position, the preferred first step in thecomputational analysis comprises the determination of the interaction ofeach possible rotamer with all or part of the remainder of the protein.That is, the energy of interaction, as measured by one or more of thescoring functions, of each possible rotamer at each variable residueposition with either the backbone or other rotamers, is calculated. In apreferred embodiment, the interaction of each rotamer with the entireremainder of the protein, i.e. both the entire template and all otherrotamers, is done. However, as outlined above, it is possible to onlymodel a portion of a protein, for example a domain of a larger protein,and thus in some cases, not all of the protein need be considered. Theterm “portion”, or similar grammatical equivalents thereof, as usedherein, with regard to a protein refers to a fragment of that protein.This fragment may range in size from 5-10 amino acid residues to theentire amino acid sequence minus one amino acid. Accordingly, the term“portion”, as used herein, with regard to a nucleic refers to a fragmentof that nucleic acid. This fragment may range in size from 6-10nucleotides to the entire nucleic acid sequence minus one nucleotide.

In a preferred embodiment, the first step of the computationalprocessing is done by calculating two sets of interactions for eachrotamer at every position: the interaction of the rotamer side chainwith the template or backbone (the “singles” energy), and theinteraction of the rotamer side chain with all other possible rotamersat every other position (the “doubles” energy), whether that position isvaried or floated. It should be understood that the backbone in thiscase includes both the atoms of the protein structure backbone, as wellas the atoms of any fixed residues, wherein the fixed residues aredefined as a particular conformation of an amino acid.

Thus, “singles” (rotamer/template) energies are calculated for theinteraction of every possible rotamer at every variable residue positionwith the backbone, using some or all of the scoring functions. Thus, forthe hydrogen bonding scoring function, every hydrogen bonding atom ofthe rotamer and every hydrogen bonding atom of the backbone isevaluated, and the E_(HB) is calculated for each possible rotamer atevery variable position. Similarly, for the van der Waals scoringfunction, every atom of the rotamer is compared to every atom of thetemplate (generally excluding the backbone atoms of its own residue),and the E_(vdW) is calculated for each possible rotamer at everyvariable residue position. In addition, generally no van der Waalsenergy is calculated if the atoms are connected by three bonds or less.For the atomic solvation scoring function, the surface of the rotamer ismeasured against the surface of the template, and the E_(as) for eachpossible rotamer at every variable residue position is calculated. Thesecondary structure propensity scoring function is also considered as asingles energy, and thus the total singles energy may contain an E_(ss)term. As will be appreciated by those in the art, many of these energyterms will be close to zero, depending on the physical distance betweenthe rotamer and the template position; that is, the farther apart thetwo moieties, the lower the energy.

For the calculation of “doubles” energy (rotamer/rotamer), theinteraction energy of each possible rotamer is compared with everypossible rotamer at all other variable residue positions. Thus,“doubles” energies are calculated for the interaction of every possiblerotamer at every variable residue position with every possible rotamerat every other variable residue position, using some or all of thescoring functions. Thus, for the hydrogen bonding scoring function,every hydrogen bonding atom of the first rotamer and every hydrogenbonding atom of every possible second rotamer is evaluated, and theE_(HB) is calculated for each possible rotamer pair for any two variablepositions. Similarly, for the van der Waals scoring function, every atomof the first rotamer is compared to every atom of every possible secondrotamer, and the E_(vdW) is calculated for each possible rotamer pair atevery two variable residue positions. For the atomic solvation scoringfunction, the surface of the first rotamer is measured against thesurface of every possible second rotamer, and the E_(as) for eachpossible rotamer pair at every two variable residue positions iscalculated. The secondary structure propensity scoring function need notbe run as a “doubles” energy, as it is considered as a component of the“singles” energy. As will be appreciated by those in the art, many ofthese double energy terms will be close to zero, depending on thephysical distance between the first rotamer and the second rotamer; thatis, the farther apart the two moieties, the lower the energy.

In addition, as will be appreciated by those in the art, a variety offorce fields that can be used in the PDA calculations can be used,including, but not limited to, Dreiding I and Dreiding II [Mayo et al,J. Phys. Chem. 94:8897 (1990)], AMBER [Weiner et al., J. Amer. Chem.Soc. 106:765 (1984) and Weiner et al., J. Comp. Chem. 106:230 (1986)],MM2 [Allinger, J. Chem. Soc. 99:8127 (1977), Liljefors et al., J. Com.Chem. 8:1051 (1987)]; MMP2 [Sprague et al., J. Comp. Chem. 8:581(1987)]; CHARMM [Brooks et al., J. Comp. Chem. 106:187 (1983)]; GROMOS;and MM3 [Allinger et al., J. Amer. Chem. Soc. 111:8551 (1989)], OPLS-AA[Jorgensen et al., J. Am. Chem. Soc. 118:11225-11236 (1996); Jorgensen,W. L.; BOSS, Version 4.1; Yale University: New Haven, Conn. (1999)];OPLS [Jorgensen et al., J. Am. Chem. Soc.110:1657ff (1988); Jorgensen etal., J Am. Chem. Soc. 112:4768ff (1990)]; UNRES (United ResidueForcefield; Liwo et al., Protein Science 2:1697-1714 (1993); Liwo etal., Protein Science 2:1715-1731 (1993); Liwo et al., J. Comp. Chem.18:849-873 (1997); Liwo et al., J. Comp. Chem. 18:874-884 (1997); Liwoet al., J. Comp. Chem. 19:259-276 (1998); Forcefield for ProteinStructure Prediction (Liwo et al., Proc. Natl. Acad. Sci. U.S.A96:5482-5485 (1999)]; ECEPP/3 [Liwo et al., J Protein Chem. 13(4):375-80(1994)]; A field (Weiner, et al., J. Am. Chem. Soc. 106:765-784); AMBER3.0 force field (U.C. Singh et al., Proc. Natl. Acad. Sci. U.S.A.82:755-759); CHARMM and CHARMM22 (Brooks et al., J. Comp. Chem.4:187-217); cvff3.0 [Dauber-Osguthorpe, et al., Proteins: Structure,Function and Genetics, 4:31-47 (1988)]; cff91 (Maple, et al., J. Comp.Chem. 15:162-182); also, the DISCOVER (cvff and cff91) and AMBERforcefields are used in the INSIGHT molecular modeling package(Biosym/MSI, San Diego Calif.) and HARMM is used in the QUANTA molecularmodeling package (Biosym/MSI, San Diego Calif.), all of which areexpressly incorporated by reference.

Once the singles and doubles energies are calculated and stored, thenext step of the computational processing may occur. As outlined in U.S.Ser. No. 09/127,926 and PCT US98/07254, preferred embodiments utilize aDead End Elimination (DEE) step, and preferably a Monte Carlo step.

PDA, viewed broadly, has three components that may be varied to alterthe output (e.g. the primary library): the scoring functions used in theprocess; the filtering technique, and the sampling technique.

In a preferred embodiment, the scoring functions may be altered. In apreferred embodiment, the scoring functions outlined above may be biasedor weighted in a variety of ways. For example, a bias towards or awayfrom a reference sequence or family of sequences can be done; forexample, a bias towards wild-type or homolog residues may be used.Similarly, the entire protein or a fragment of it may be biased; forexample, the active site may be biased towards wild-type residues, ordomain residues towards a particular desired physical property can bedone. Furthermore, a bias towards or against increased energy can begenerated. Additional scoring function biases include, but are notlimited to applying electrostatic potential gradients or hydrophobicitygradients, adding a substrate or binding partner to the calculation, orbiasing towards a desired charge or hydrophobicity.

In addition, in an alternative embodiment, there are a variety ofadditional scoring functions that may be used. Additional scoringfunctions include, but are not limited to torsional potentials, orresidue pair potentials, or residue entropy potentials. Such additionalscoring functions can be used alone, or as functions for processing thelibrary after it is scored initially. For example, a variety offunctions derived from data on binding of peptides to MHC (MajorHistocompabbility Complex) can be used to rescore a library in order toeliminate proteins containing sequences which can potentially bind toMHC, i.e. potentially immunogenic sequences.

In a preferred embodiment, a variety of filtering techniques can bedone, including, but not limited to, DEE and its related counterparts.Additional filtering techniques include, but are not limited tobranch-and-bound techniques for finding optimal sequences (Gordon andMayo, Structure Fold. Des. 7:1089-98, 1999), and exhaustive enumerationof sequences.

As will be appreciated by those in the art, once an optimized sequenceor set of sequences is generated, a variety of sequence space samplingmethods can be done, either in addition to the preferred Monte Carlomethods, or instead of a Monte Carlo search. That is, once a sequence orset of sequences is generated, preferred methods utilize samplingtechniques to allow the generation of additional, related sequences fortesting.

These sampling methods can include the use of amino acid substitutions,insertions or deletions, or recombinations of one or more sequences. Asoutlined herein, a preferred embodiment utilizes a Monte Carlo search,which is a series of biased, systematic, or random jumps. However, thereare other sampling techniques that can be used, including Boltzmansampling, genetic algorithm techniques and simulated annealing. Inaddition, for all the sampling techniques, the kinds of jumps allowedcan be altered (e.g. random jumps to random residues, biased jumps (toor away from wild-type, for example), jumps to biased residues (to oraway from similar residues, for example, etc.). Jumps where multipleresidue positions are coupled (two residues always change together, ornever change together), jumps where whole sets of residues change toother sequences (e.g., recombination). Similarly, for all the samplingtechniques, the acceptance criteria of whether a sampling jump isaccepted can be altered.

In addition, it should be noted that the preferred methods of theinvention result in a rank ordered list of sequences; that is, thesequences are ranked on the basis of some objective criteria. However,as outlined herein, it is possible to create a set of non-orderedsequences, for example by generating a probability table directly (forexample using SCMF analysis or sequence alignment techniques) that listssequences without ranking them. The sampling techniques outlined hereincan be used in either situation.

In a preferred embodiment, Boltzman sampling is done. As will beappreciated by those in the art, the temperature criteria for Boltzmansampling can be altered to allow broad searches at high temperature andnarrow searches close to local optima at low temperatures (see e.g.,Metropolis et al., J. Chem. Phys. 21:1087, 1953).

In a preferred embodiment, the sampling technique utilizes geneticalgorithms, e.g., such as those described by Holland (Adaptation inNatural and Artifical Systems, 1975, Ann Arbor, U. Michigan Press).Genetic algorithm analysis generally takes generated sequences andrecombines them computationally, similar to a nucleic acid recombinationevent, in a manner similar to “gene shuffling”. Thus the “jumps” ofgenetic algorithm analysis generally are multiple position jumps. Inaddition, as outlined below, correlated multiple jumps may also be done.Such jumps can occur with different crossover positions and more thanone recombination at a time, and can involve recombination of two ormore sequences. Furthermore, deletions or insertions (random or biased)can be done. In addition, as outlined below, genetic algorithm analysismay also be used after the secondary library has been generated.

In a preferred embodiment, the sampling technique utilizes simulatedannealing, e.g., such as described by Kirkpatrick et al. [Science,220:671-680 (1983)]. Simulated annealing alters the cutoff for acceptinggood or bad jumps by altering the temperature. That is, the stringencyof the cutoff is altered by altering the temperature. This allows broadsearches at high temperature to new areas of sequence space, alteringwith narrow searches at low temperature to explore regions in detail.

In addition, as outlined below, these sampling methods can be used tofurther process a first set to generate additional sets of IbA proteins.

The computational processing results in a set of optimized IbA proteinsequences. These optimized IbA protein sequences are generallysignificantly different from the wild-type IFN-β sequence from which thebackbone was taken. That is, each optimized IbA protein sequencepreferably comprises at least about 3-10% variant amino acids from thestarting or wild type sequence, with at least about 10-15% beingpreferred, with at least about 15-20% changes being more preferred andat least 25% being particularly preferred.

In a preferred embodiment, the IbA proteins of the invention have 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or40 different residues from the human IFN-β sequence.

Thus, in the broadest sense, the present invention is directed to IbAproteins that have IFN-β activity. By “IFN-β activity” or “IbA” hereinis meant that the IbA protein exhibits at least one, and preferablymore, of the biological functions of an IFN-β, as defined below. In oneembodiment, the biological function of an IbA protein is altered,preferably improved, over the corresponding biological activity of anIFN-β.

By “protein” herein is meant at least two covalently attached aminoacids, which includes proteins, polypeptides, oligopeptides andpeptides. The protein may be made up of naturally occurring amino acidsand peptide bonds, or synthetic peptidomimetic structures, i.e.,“analogs” such as peptoids [see Simon et al., Proc. Natl. Acd. Sci.U.S.A. 89(20:9367-71 (1992)], generally depending on the method ofsynthesis. Thus “amino acid”, or “peptide residue”, as used herein meansboth naturally occurring and synthetic amino acids. For example,homo-phenylalanine, citrulline, and noreleucine are considered aminoacids for the purposes of the invention. “Amino acid” also includesamino acid residues such as proline and hydroxyproline. In addition, anyamino acid representing a component of the IbA proteins can be replacedby the same amino acid but of the opposite chirality. Thus, any aminoacid naturally occurring in the L-conflguration (which may also bereferred to as the R or S, depending upon the structure of the chemicalentity) may be replaced with an amino acid of the same chemicalstructural type, but of the opposite chirality, generally referred to asthe D- amino acid but which can additionally be referred to as the R- orthe S-, depending upon its composition and chemical configuration. Suchderivatives have the property of greatly increased stability, andtherefore are advantageous in the formulation of compounds which mayhave longer in vivo half lives, when administered by oral, intravenous,intramuscular, intraperitoneal, topical, rectal, intraocular, or otherroutes. In the preferred embodiment, the amino acids are in the (S) orL-configuration. If non-naturally occurring side chains are used,non-amino acid substituents may be used, for example to prevent orretard in vivo degradations. Proteins including non-naturally occurringamino acids may be synthesized or in some cases, made recombinantly; seevan Hest et al., FEBS Left 428:(1-2) 68-70 May 22, 1998 and Tang et al.,Abstr. Pap Am. Chem. S218:U138-U138 Part 2 Aug. 22, 1999, both of whichare expressly incorporated by reference herein.

Additionally, modified amino acids or chemical derivatives of aminoacids of consensus or fragments of IbA proteins, according to thepresent invention may be provided, which polypeptides contain additionalchemical moieties or modified amino acids not normally a part of theprotein. Covalent and non-covalent modifications of the protein are thusincluded within the scope of the present invention.

Such modifications may be introduced into an IbA polypeptide by reactingtargeted amino acid residues of the polypeptide with an organicderivatizing agent that is capable of reacting with selected side chainsor terminal residues. The following examples of chemical derivatives areprovided by way of illustration and not by way of limitation.

Aromatic amino acids may be replaced with D- or L-naphylalanine, D- orL-Phenylglycine, D- or L-2-thieneylalanine, D- or L-1-, 2-, 3- or4-pyreneylalanine, D- or L-3-thieneylalanine, D- orL-(2-pyridinyl)-alanine, D- or L-(3-pyridinyl)-alanine, D- orL-(2-pyrazinyl)-alanine, D- or L-(4-isopropyl)-phenylglycine,D-(trifluoromethyl)-phenylglycine, D-(trifluoromethyl)-phenylalanine,D-p-fluorophenylalanine, D- or L-p-biphenylphenylalanine, D- orL-p-methoxybiphenylphenylalanine, D- or L-2-indole(alkyl)alanines, andD- or L-alkylainines where alkyl may be substituted or unsubstitutedmethyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl,sec-isotyl, iso-pentyl, non-acidic amino acids, of C1-C20.

Acidic amino acids can be substituted with non-carboxylate amino acidswhile maintaining a negative charge, and derivatives or analogs thereof,such as the non-limiting examples of (phosphono)alanine,(phosphono)glycine, (phosphono)leucine, (phosphono)isoleucine,(phosphono)threonine, or (phosphono)senne; or sulfated (e.g., —SO₃H)threonine, serine, tyrosine.

Other substitutions may include unnatural hyroxylated amino acids thatmay be made by combining “alkyl” with any natural amino acid. The term“alkyl” as used herein refers to a branched or unbranched saturatedhydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl,n-propyl, isoptopyl, n-butyl, isobutyl, t-butyl, octyl, decyl,tetradecyl, hexadecyl, eicosyl, tetracisyl and the like. Preferred alkylgroups herein contain 1 to 12 carbon atoms. Also included within thedefinition of an alkyl group are cycloalkyl groups such as C5 and C6rings, and heterocyclic rings with nitrogen, oxygen, sulfur orphosphorus. Alkyl also includes heteroalkyl, with heteroatoms of sulfur,oxygen, and nitrogen being preferred. Alkyl includes substituted alkylgroups. By “substituted alkyl group” herein is meant an alkyl groupfurther comprising one or more substitution moieties. A preferredheteroalkyl group is an alkyl amine. By “alkyl amine” or grammaticalequivalents herein is meant an alkyl group as defined above, substitutedwith an amine group at any position. In addition, the alkyl amine mayhave other substitution groups, as outlined above for alkyl group. Theamine may be primary (—NH₂R), secondary (—NHR), or tertiary (—NR₃).Basic amino acids may be substituted with alkyl groups at any positionof the naturally occurring amino acids lysine, arginine, ornithine,citrulline, or (guanidino)-acetic acid, or other (guanidino)alkyl-aceticacids, where “alkyl” is define as above. Nitrile derivatives (e.g.,containing the CN-moiety in place of COOH) may also be substituted forasparagine or glutamine, and methionine sulfoxide may be substituted formethionine. Methods of preparation of such peptide derivatives are wellknown to one skilled in the art.

In addition, any amide linkage in any of the IbA polypeptides can bereplaced by a ketomethylene moiety. Such derivatives are expected tohave the property of increased stability to degradation by enzymes, andtherefore possess advantages for the formulation of compounds which mayhave increased in vivo half lives, as administered by oral, intravenous,intramuscular, intraperitoneal, topical, rectal, intraocular, or otherroutes.

Additional amino acid modifications of amino acids of IbA polypeptidesof the present invention may include the following: Cysteinyl residuesmay be reacted with alpha-haloacetates (and corresponding amines), suchas 2-chloroacetic acid or chloroacetamide, to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteinyl residues may also bederivatized by reaction with compounds such as bromotrifluoroacetone,alpha-bromo-beta-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues may be derivatzed by reaction with compounds such asdiethylprocarbonate e.g., at pH 5.5-7.0 because this agent is relativelyspecific for the histidyl side chain, and para-bromophenacyl bromide mayalso be used; e.g., where the reaction is preferably performed in 0.1Msodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues may be reacted with compounds suchas succinic or other carboxylic acid anhydrides. Derivatization withthese agents is expected to have the effect of reversing the charge ofthe lysinyl residues. Other suitable reagents for derivatizingalpha-amino-containing residues include compounds such asimidoesters/e.g., as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues may be modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin according to known method steps.Derivatization of arginine residues requires that the reaction beperformed in alkaline conditions because of the high pKa of theguanidine functional group. Furthermore, these reagents may react withthe groups of lysine as well as the arginine epsilon-amino group.

The specific modification of tyrosyl residues per se is well-known, suchas for introducing spectral labels into tyrosyl residues by reactionwith aromatic diazonium compounds or tetranitromethane. N-acetylimidizoland tetranitromethane may be used to form O-acetyl tyrosyl species and3-nitro derivatives, respectively.

Carboxyl side groups (aspartyl or glutamyl) may be selectively modifiedby reaction with carbodiimides (R′-N-C-N-R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermoreaspartyl and glutamyl residues may be converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues may be frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues may be deamidated under mildly acidic conditions. Either formof these residues falls within the scope of the present invention.

The IFN-β may be from any number of organisms, with IFN-β s from mammalsbeing particularly preferred. Suitable mammals include, but are notlimited to, rodents (rats, mice, hamsters, guinea pigs, etc.), primates,farm animals (including sheep, goats, pigs, cows, horses, etc) and inthe most preferred embodiment, from humans (this is sometimes referredto herein as hIFN-β, the sequence of which is depicted in FIG. 1). Aswill be appreciated by those in the art, IFN-β s based on IFN-β s frommammals other than humans may find use in animal models of humandisease. The GenBank accession numbers for a variety of mammalian IFN-βspecies is as follows: bovine 69689, 124465 (IFN-β-1 precursor), 69688,124467 (IFN-β-3 precursor), 69687, 124466 (IFN-β-2 precursor); dog442673; sheep 310382; cat CAA69853, 1754718; pig 2411469, 164517; mouse69686, 6754304, 51551, 124470, 494203; rat 7438651, 2497434, 1616939;Macaca fascicularis 3766295; horse 69685, 124468, 164229; human 69684,124469, 4504603, 3318961, 3318960.

The IbA proteins of the invention exhibit at least one biologicalfunction of an IFN-β. By “interferon-beta” or “IFN-β” herein is meant awild type IFN-β or an allelic variant thereof. Thus, IFN-β refers to allforms of IFN-β that are active in accepted IFN-β assays.

The IbA proteins of the invention exhibit at least one biologicalfunction of an IFN-β. By “biological function” or “biological property”herein is meant any one of the properties or functions of an IFN-β,including, but not limited to, the ability to effect cellular growth, inparticular inhibition of cell proliferation; the ability to effectcellular differentiation, in particular induction of celldifferentiation; the ability to induce changes in cell morphology; theability to modulate the immune system; the ability to enhancehistocompafibility antigen expression; the ability to stimulateimmunoglobulin-Fc receptor expression on macrophages; the ability toinduce antibody production in B lymphocytes, the ability to activatenatural killer cells; the ability to bind to an IFN receptor; theability to bind to a cell comprising an IFN receptor, the ability totreat multiple sclerosis; the ability to treat idiopathic pulmonaryfibrosis; the ability to treat inflammatory diseases; the ability totreat viral diseases, including treatment of infections caused bypapilloma viruses, such as genital warts and condylomata of the uterinecervix; hepatitis viruses, such as acute/chronic hepatitis B and non-A,non-B hepatitis (hepatitis C); herpes viruses, such as herpes genitalis,herpes zoster, herpes keratitis, and herpes simplex; viral encephalitis;cytomegalovirus pneumonia; and prophylaxis of rhinovirus; the ability totreat cancer, including treatment of several malignant diseases such asosteosarcoma, basal cell carcinoma, cervical dysplasia, glioma, acutemyeloid leukemia, multiple myeloma, Hodgkin's disease, melanoma, renalcancer, liver cancer, and breast cancer.

All of these IbA proteins will exhibit at least 50% of the receptorbinding or biological activity as the wild type IFN-β. More preferredare IbA proteins that exhibit at least 75%, even more preferred are IbAproteins that exhibit at least 90%, and most preferred are IbA proteinsthat exhibit more than 100% of the receptor binding or biologicalactivity as the wild type IFN-β. Biological assays, receptor bindingassays, anti-viral and anti-proliferation assays are described in U.S.patents 4,450,103; 4,518,584; 4,588,585; 4,737,462; 4,738,844;4,738,845; 4,753,795; 4,769,233; 4,793,995; 4,914,033; 4,959,314;5,183,746; 5,376,567; 5,545,723; 5,730,969; 5,814,485; 5,869,603 and ine.g., Anderson et al., J. Biol. Chem. 257(19):11301-4 (1982); Herbermanet al., Nature 277(5693):221-3 (1979); Williams et al., Nature282(5739):582-6 (1979); Branca and Baglioni, Nature 294(5843):768-70(1981); Proc. Natl. Acad. Sci. U.S.A. 81(18):5662-6 (1984); Fellous etal., Proc. Natl. Acad. Sci. U.S.A. 79(10):3082-6 (1982); and Runkel etal., J. Biol. Chem. 273(14):8003-8 (1998), all of which are expresslyincorporated by reference.

In one embodiment, at least one biological property of the IbA proteinis altered when compared to the same property of IFN-β. As outlinedabove, the invention provides IbA nucleic acids encoding IbApolypeptides. The IbA polypeptide preferably has at least one property,which is substantially different from the same property of thecorresponding naturally occurring IFN-β polypeptide. The property of theIbA polypeptide is the result the PDA analysis of the present invention.

The term “altered property” or grammatical equivalents thereof in thecontext of a polypeptide, as used herein, refer to any characteristic orattribute of a polypeptide that can be selected or detected and comparedto the corresponding property of a naturally occurring protein. Theseproperties include, but are not limited to oxidative stability,substrate specificity, substrate binding or catalytic activity, thermalstability, alkaline stability, pH activity profile, resistance toproteolytic degradation, Km, kcat, Km/kcat ratio, kinetic association(K_(on)) and dissociation (K_(off)) rate, protein folding, inducing animmune response, ability to bind to a ligand, ability to bind to areceptor, ability to be secreted, ability to be displayed on the surfaceof a cell, ability to oligomerize, ability to signal, ability tostimulate cell proliferation, ability to inhibit cell proliferation,ability to induce apoptosis, ability to be modified by phosphorylabon orglycosylation, ability to treat disease.

Unless otherwise specified, a substantial change in any of theabove-listed properties, when comparing the property of an IbApolypeptide to the property of a naturally occurring IFN-β protein ispreferably at least a 20%, more preferably, 50%, more preferably atleast a 2-fold increase or decrease.

A change in oxidative stability is evidenced by at least about 20%, morepreferably at least 50% increase of activity of an IbA protein whenexposed to various oxidizing conditions as compared to that of IFN-β.Oxidative stability is measured by known procedures.

A change in alkaline stability is evidenced by at least about a 5% orgreater increase or decrease (preferably increase) in the half life ofthe activity of an IbA protein when exposed to increasing or decreasingpH conditions as compared to that of IFN-β. Generally, alkalinestability is measured by known procedures.

A change in thermal stability is evidenced by at least about a 5% orgreater increase or decrease (preferably increase) in the half life ofthe activity of an IbA protein when exposed to a relatively hightemperature and neutral pH as compared to that of IFN-β. Generally,thermal stability is measured by known procedures.

Similarly, IbA proteins, for example are experimentally tested andvalidated in in vivo and in in vitro assays. Suitable assays include,but are not limited to, e.g., examining their binding affinity tonatural occurring or variant receptors and to high affinity agonistsand/or antagonists. In addition to cell-free biochemical affinity tests,quantitative comparison are made comparing kinetic and equilibriumbinding constants for the natural receptor to the naturally occurringIFN-β and to the IbA proteins. The kinetic association rate (K_(on)) anddissociation rate (K_(off)), and the equilibrium binding constants(K_(d)) can be determined using surface plasmon resonance on a BlAcoreinstrument following the standard procedure in the literature [Pearce etal., Biochemistry 38:81-89 (1999)]. Comparing the binding constantbetween a natural receptor and its corresponding naturally occurringIFN-β with the binding constant of a natural occurring receptor and anIbA protein are made in order to evaluate the sensitivity andspecificity of the IbA protein. Preferably, binding affinity of the IbAprotein to natural receptors and agonists increases relative to thenaturally occurring IFN-β, while antagonist affinity decreases. IbAproteins with higher affinity to antagonists relative to the IFN-β mayalso be generated by the methods of the invention.

As described above, one biological function of an IbA protein is theability of the IbA protein to bind to cells comprising an interferonreceptor.

In a preferred embodiment, the assay system used to determine IbA is anin vitro system using cells that either express endogenous interferonreceptors or cells stably transfected with the gene encoding the humaninterferon receptor. In this system, cell proliferation is measured as afunction of BrdU incorporation, which is incorporated into the nucleicacid of proliferating cells. A decrease above background of at leastabout 10%, with at least about 20% being preferred, with at least about30% being more preferred and at least about 50%, 75% and 90% beingespecially preferred is an indication of IbA.

In a preferred embodiment, the antigenic profile in the host animal ofthe IbA protein is similar, and preferably identical, to the antigenicprofile of the host IFN-β; that is, the IbA protein does notsignificantly stimulate the host organism (e.g. the patient) to animmune response; that is, any immune response is not clinically relevantand there is no allergic response or neutralization of the protein by anantibody. That is, in a preferred embodiment, the IbA protein does notcontain additional or different epitopes from the IFN-β. By “epitope” or“determinant” herein is meant a portion of a protein which will generateand/or bind an antibody. Thus, in most instances, no significant amountof antibodies are generated to a IbA protein. In general, this isaccomplished by not significantly altering surface residues, as outlinedbelow nor by adding any amino acid residues on the surface which canbecome glycosylated, as novel glycosylation can result in an immuneresponse.

The IbA proteins and nucleic acids of the invention are distinguishablefrom naturally occurring IFN-ps. By “naturally occurring” or “wild type”or grammatical equivalents, herein is meant an amino acid sequence or anucleotide sequence that is found in nature and includes allelicvariations; that is, an amino acid sequence or a nucleotide sequencethat usually has not been intentionally modified. Accordingly, by“non-naturally occurring” or “synthetic” or “recombinant” or grammaticalequivalents thereof, herein is meant an amino acid sequence or anucleotide sequence that is not found in nature; that is, an amino acidsequence or a nucleotide sequence that usually has been intentionallymodified. It is understood that once a recombinant nucleic acid is madeand reintroduced into a host cell or organism, it will replicatenon-recombinantly, i.e., using the in vivo cellular machinery of thehost cell rather than in vitro manipulations, however, such nucleicacids, once produced recombinantly, although subsequently replicatednon-recombinantly, are still considered recombinant for the purpose ofthe invention. Representative amino acid and nucleotide sequences of anaturally occurring human IFN-β are shown in FIG. 1. It should be notedthat unless otherwise stated, all positional numbering of IbA proteinsand IbA nucleic acids is based on these sequences. That is, as will beappreciated by those in the art, an alignment of IFN-β proteins and IbAproteins can be done using standard programs, as is outlined below, withthe identification of “equivalent” positions between the two proteins.Thus, the IbA proteins and nucleic acids of the invention arenon-naturally occurring; that is, they do not exist in nature.

Thus, in a preferred embodiment, the IbA protein has an amino acidsequence that differs from a wild-type IFN-β sequence by at least 3% ofthe residues. That is, the IbA proteins of the invention are less thanabout 97% identical to an IFN-β amino acid sequence. Accordingly, aprotein is an “IbA protein” if the overall homology of the proteinsequence to the amino acid sequence shown in FIG. 1A or FIG. 1B (SEQ IDNO:1) is preferably less than about 97%, more preferably less than about95%, even more preferably less than about 90% and most preferably lessthan 85%. In some embodiments the homology will be as low as about 75 to80%. Stated differently, based on the human IFN-β sequence of 166residues (see FIG. 1A) (SEQ ID NO:1), IbA proteins have at least about 5residues that differ from the human IFN-β sequence (3%), with IbAproteins having from 5 residues to upwards of 62 residues beingdifferent from the human IFN-β sequence. Preferred IbA proteins have5-30 different residues with from about 5 to about 15 being particularlypreferred (that is, 3-9% of the protein is not identical to humanIFN-β).

In another preferred embodiment, IbA proteins have 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 differentresidues from the human IFN-β sequence.

Homology in this context means sequence similarity or identity, withidentity being preferred. As is known in the art, a number of differentprograms can be used to identify whether a protein (or nucleic acid asdiscussed below) has sequence identity or similarity to a knownsequence. Sequence identity and/or similarity is determined usingstandard techniques known in the art, including, but not limited to, thelocal sequence identity algorithm of Smith & Waterman, Adv. Appl. Math.,2:482 (1981), by the sequence identity alignment algorithm of Needleman& Wunsch, J. Mol. Biol., 48:443 (1970), by the search for similaritymethod of Pearson & Lipman, Proc. Natl. Acad. Sci. U.S.A., 85:2444(1988), by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Drive, Madison, Wis.), the Best Fitsequence program described by Devereux et al., Nucl. Acid Res.,12:387-395 (1984), preferably using the default settings, or byinspection. Preferably, percent identity is calculated by FastDB basedupon the following parameters: mismatch penalty of 1; gap penalty of 1;gap size penalty of 0.33; and joining penalty of 30, “Current Methods inSequence Comparison and Analysis,” Macromolecule Sequencing andSynthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R.Liss, Inc.

An example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Dooliftle, J. Mol. Evol.35:351-360 (1987); the method is similar to that described by Higgins &Sharp CABIOS 5:151-153 (1989). Useful PILEUP parameters including adefault gap weight of 3.00, a default gap length weight of 0.10, andweighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, describedin: Altschul et al., J. Mol. Biol. 215, 403-410, (1990); Altschul etal., Nucleic Acids Res. 25:3389-3402 (1997); and Karlin et al., Proc.Natl. Acad. Sci. U.S.A. 90:5873-5787 (1993). A particularly useful BLASTprogram is the WU-BLAST-2 program which was obtained from Altschul etal., Methods in Enzymology, 266:460-480 (1996);http:flblast.wustl/edu/blastt README.html]. WU-BLAST-2 uses severalsearch parameters, most of which are set to the default values. Theadjustable parameters are set with the following values: overlap span=1,overlap fraction=0.125, word threshold (T)=11. The HSP S and HSP S2parameters are dynamic values and are established by the program itselfdepending upon the composition of the particular sequence andcomposition of the particular database against which the sequence ofinterest is being searched; however, the values may be adjusted toincrease sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschulet al., Nucl. Acids Res., 25:3389-3402. Gapped BLAST uses BLOSUM-62substitution scores; threshold T parameter set to 9; the two-hit methodto trigger ungapped extensions; charges gap lengths of k a cost of 10+k;X_(u) set to 16, and X_(g) set to 40 for database search stage and to 67for the output stage of the algorithms. Gapped alignments are triggeredby a score corresponding to ˜22 bits.

A % amino acid sequence identity value is determined by the number ofmatching identical residues divided by the total number of residues ofthe “longer” sequence in the aligned region. The “longer” sequence isthe one having the most actual residues in the aligned region (gapsintroduced by WU-Blast-2 to maximize the alignment score are ignored).

In a similar manner, “percent (%) nucleic acid sequence identity” withrespect to the coding sequence of the polypeptides identified herein isdefined as the percentage of nucleotide residues in a candidate sequencethat are identical with the nucleofide residues in the coding sequenceof the cell cycle protein. A preferred method utilizes the BLASTN moduleof WU-BLAST-2 set to the default parameters, with overlap span andoverlap fraction set to 1 and 0.125, respectively.

The alignment may include the introduction of gaps in the sequences tobe aligned. In addition, for sequences which contain either more orfewer amino acids than the protein encoded by the sequence of FIG. 1, itis understood that in one embodiment, the percentage of sequenceidentity will be determined based on the number of identical amino acidsin relation to the total number of amino acids. Thus, for example,sequence identity of sequences shorter than that shown in FIG. 1, asdiscussed below, will be determined using the number of amino acids inthe shorter sequence, in one embodiment. In percent identitycalculations relative weight is not assigned to various manifestationsof sequence variation, such as, insertions, deletions, substitutions,etc.

In one embodiment, only identities are scored positively (+1) and allforms of sequence variation including gaps are assigned a value of “0”,which obviates the need for a weighted scale or parameters as describedbelow for sequence similarity calculations. Percent sequence identitycan be calculated, for example, by dividing the number of matchingidentical residues by the total number of residues of the “shorter”sequence in the aligned region and multiplying by 100. The “longer”sequence is the one having the most actual residues in the alignedregion.

Thus, IbA proteins of the present invention may be shorter or longerthan the amino acid sequence shown in FIG. 1A (SEQ ID NO:1). Thus, in apreferred embodiment, included within the definition of IbA proteins areportions or fragments of the sequences depicted herein. Fragments of IbAproteins are considered IbA proteins if a) they share at least oneantigenic epitope; b) have at least the indicated homology; c) andpreferably have IbA biological activity as defined herein.

In a preferred embodiment, as is more fully outlined below, the IbAproteins include further amino acid variations, as compared to a wildtype IFN-β, than those outlined herein. In addition, as outlined herein,any of the variations depicted herein may be combined in any way to formadditional novel IbA proteins.

In addition, IbA proteins can be made that are longer than thosedepicted in the figures, for example, by the addition of epitope orpurification tags, as outlined herein, the addition of other fusionsequences, etc. For example, the IbA proteins of the invention may befused to other therapeutic proteins such as IL-11 or to other proteinssuch as Fc or serum albumin for pharmacokinetic purposes. See forexample U.S. Pat. No. 5,766,883 and 5,876,969, both of which areexpressly incorporated by reference.

In a preferred embodiment, the IbA proteins comprise variable residuesin core residues.

Human IFN-β core residues are as follows: positions 1, 6, 10, 13, 14,15, 17, 18, 21, 38, 50, 55, 56, 58, 59, 61, 62, 63, 66, 69, 70, 72, 74,76, 77, 81, 84, 87, 90, 91, 94, 95, 98, 102, 114, 115, 118, 122, 125,126, 129, 130, 132, 133, 136, 138, 139, 142, 143, 144, 146, 147, 150,151, 153, 154, 157, 159, 160, 161, 163, and 164 (see FIG. 3).Accordingly, in a preferred embodiment, IbA proteins have variablepositions selected from these positions.

The structure of human IFN-β as reported by Karpasus et al. (supra)indicated that IFN-β forms a dimer consisting of an A-chain and aB-chain.

Thus, in one embodiment, variable residues for the A-chain are asfollows: positions 1, 6, 10, 13, 14, 17, 18, 21, 38, 50, 55, 56, 58, 59,61, 62, 63, 66, 69, 70, 72, 74, 76, 77, 81, 84, 87, 90, 91, 94, 95, 98,102, 114, 115, 118, 122, 125, 126, 129, 130, 132, 133, 136, 138, 139,142, 143, 144, 146, 147, 150, 151, 153, 154, 157, 159, 160, 161, 163,and 164 (see FIG. 3). Accordingly, in a preferred embodiment, IbAproteins have variable positions selected from these positions.

Thus, in another embodiment, variable residues for the B-chain are asfollows: positions 1, 6, 10, 13, 14, 15, 17, 18, 21, 38, 50, 55, 56, 58,59, 61, 62, 63, 66, 69, 70, 72, 74, 76, 77, 81, 84, 87, 90, 91, 94, 95,98, 102, 114, 115, 118, 122, 125, 126, 129, 130, 132, 133, 136, 138,139, 142, 143, 144, 146, 147, 150, 151, 153, 154, 157, 159, 160, 161,163, and 164 (see FIG. 3). Accordingly, in a preferred embodiment, IbAproteins have variable positions selected from these positions.

In a preferred embodiment, IbA proteins have variable positions selectedsolely from core residues of human IFN-β. Alternatively, at least amajority (51%) of the variable positions are selected from coreresidues, with at least about 75% of the variable positions beingpreferably selected from core residue positions, and at least about 90%of the variable positions being particularly preferred. A specificallypreferred embodiment has only core variable positions altered ascompared to human IFN-β.

Particularly preferred embodiments where IbA proteins have variable corepositions as compared to human IFN-β are shown in the Figures.

In one embodiment, the variable core positions are altered to any of theother 19 amino acids. In a preferred embodiment, the variable coreresidues are chosen from Ala, Val, Phe, Ile, Leu, Tyr, Trp and Met. Inanother preferred embodiment, the variable core residues are chosen fromAla, Val, Leu, Ile, Phe, Tyr, and Trp. In another preferred embodiment,the variable core residues are chosen from Ala, Val, Ieu, Ile, and Gly.In another preferred embodiment, the variable core residues are chosenfrom Ala, Gly, Ser, Thr, Glu, Asp, Gln, Asn, and Cys.

In a preferred embodiment, the IbA protein of the invention has asequence that differs from a wild-type human IFN-β protein in at leastone amino acid position selected from positions 6, 13, 17, 21, 56, 3059, 61, 62, 63, 66, 69, 84, 87, 91, 98, 102, 114, 118, 122, 129, 146,150, 154, 157, 160, and 161; see also FIG. 3, which outlines sets ofamino acid positions.

Preferred amino acids for each position, including the human IFN-βresidues, are shown in FIGS. 4-16 (SEQ ID NOS:4-24). Thus, for example,for the A-chain of an IbA protein, at position 13, preferred amino acidsare Phe, Tyr, Glu, and Ala; at position 17, a preferred amino acid isAsp; at position 69, a preferred amino acid is Val; at position 84 apreferred amino acid is lie; at position 87, a preferred amino acid isPhe; at position 91, a preferred amino acid is lie; at position 98, apreferred amino acid is Phe; at position 118, preferred amino acids areAla, Val, and Cys; at position 122, preferred amino acids are Ile andVal; at position 146, a preferred amino acid is Ile; at position 157, apreferred amino acid is Leu; and at position 161, preferred amino acidsare Ala and Cys.

For the B-chain of an IbA protein, at position13, preferred amino acidsare Leu and Glu; at position 17, preferred amino acid are Ala and Thr;at position 56, a preferred amino acid is Leu; at position 63, apreferred amino acid is Phe; at position 84 a preferred amino acid islie; at position 87, a preferred amino acid is Phe; at position 91, apreferred amino acid is Ile; at position 114, preferred amino acids arePhe and Leu; at position 118, preferred amino acids are Leu and Glu; atposition 122, preferred amino acids are Ile and Phe; and at position161, preferred amino acids are Ala and Glu. Preferred changes are asfollows: L6A; L6F; S13F; S13Y; S13L; S131; S13A; S13G; S13G; S13T; S13C;S13E; C17A; C17L; C17V; C17D; C17T; C171; C17E; C17S; C17G; L211; L21V;L21A; L21Y; L21F; A56L; 159V; 159A; 159L; M621; M62V; M62L; L63A; L63F;L63Y; I66L; I66V; I66A; I69V; I69L; I69A; V84I; V84L; V84A; L87F; L87I;L87Y; L87V; L87A; L87W; V91I; V91A; V91L; V91F; V91Y; V98A; L98F; G114F;G114L; S118A; S118V; S118C; S118L; S118E; L122I; L122V; L122A; L122F;L122Y; L122W; I129V; I129L; I129A; V146I; V146A; I150V; I150A; I150L;I150F; F154L; F154Y; F157V; I157V; I157L; I157A; L160I; L160V; L160A;L160F; L160Y; T115A; T161V; T161I; T161D; T161C; T161E; and T161G. Thesemay be done either individually or in combination, with any combinationbeing possible. However, as outlined herein, preferred embodimentsutilize at least five, and preferably more, variable positions in eachIbA protein.

Particularly preferred sequences for IbA proteins are selected from thegroup consisting of: [V841 and L87F (FIG. 4B and FIG. 10B) (SEQ IDNOS:4,18)]; [V84I, V91I, L98F, L122I, and I157L (see FIG. 5B) (SEQ IDNO:5)]; [S13F, I69V, V84I, V91I, L98F, S118A, L122I, V146I, I157L, andT161A (see FIG. 6B) (SEQ ID NO:6)]; [S13Y, I69V, V84I, V91I, L98F,S118V, L122V, V146I, I157L, and T161A (see FIG. 6C) (SEQ ID NO:7)];[S13F, V84I, V91I, L98F, S118A, L122I, I157L, and T161A (see FIG. 6D)(SEQ ID NO:8)]; [S13F, C17D, I69V, V84I, V91I, L98F, S118A, L122I,VI146I, I157L, and T161A (see FIG. 7B) (SEQ ID NO:9)]; [S13Y, C17D,169V, V841, V91I, L98F, S118V, L122A, V146I, I157L, and T161A (see FIG.7C) (SEQ ID NO:10)]; [S13F, C17D, V84I, V91I, L98F, S118A, L122I, I157L,and T161A (see FIG. 70) (SEQ ID NO:11)]; [S13E, C17D, V84I, V91I, S118C,V146I, and T161C (see FIG. 8B) (SEQ ID NO:12)]; [S13A, V84I, V91I,S118C, V146I, I157L, and T161C (see FIG. 8C) (SEQ ID NO:13)]; [S13E,C17D, V84I, V91I, S118C, and T161C (see FIG. 8D)(SEQ ID NO:14)]; [S13E,C17D, 169V, V84I, V91I, S118A, L122I, V146I, I157L, and T161(see FIG.9B) (SEQ ID NO:15)]; [S13E, C17D, V84I, V91I, S118A, V146I, and 1157L(see FIG. 9C) (SEQ ID NO:16)]; [S13E, C17D, V84I, V91I, S118A, L122I,I157L, and T161A (see FIG. 9D) (SEQ ID NO:17)]; [A56L, L63F, V84I, L87F,V91I, and L122F (see FIG. 11B) (SEQ ID NO:19)]; [S13L, A56L, V84I, V91I,G114F, S118L, L122I, and T161A (see FIG. 12B) (SEQ ID NO:20)]; [S13L,C17A, A56L, V84I, L87F, V91L, G114F, S118L, L122I, and T161E (see FIG.13B) (SEQ ID NO:21)]; [S13E, A56L, V84I, V91I, G114L, S118E, and T161E(see FIG. 14B) (SEQ ID NO:22)]; [C17T, A56L, V84I, V84I, V91I, S118E,G114L, S118E, and T161E (see FIG. 15B) (SEQ ID NO:23)]; and [C17T, A56L,V84I, V91I, S118E, and T161E (see FIG. 16B) (SEQ ID NO:24)].

Particularly preferred sequences for the A-chain of an IbA protein areselected from the group consisting of: [V84I and L87F (FIG. 4B) (SEQ IDNO:4)]; [V84I, V91I, L98F, L122I, and I157L (see FIG. 5B) (SEQ IDNO:5)]; [S13F, I69V, V84I, V91I, L98F, S118A, L122I, V146I, I157L, andT161A (see FIG. 6B)(SEQ ID NO:6)]; [S13Y, I69V, V84I, V91I, L98F, S118V,L122V, V146I, I157L, and T161A (see FIG. 6C) (SEQ ID NO:7)]; [S13F,V84I, V91I, L98F, S118A, L122I, I157L, and T161A (see FIG. 6D) (SEQ IDNO:8)]; [S13F, C17D, I69V, V84I, V91I, L98F, S118A, L122I, V146I, I157L,and T161A (see FIG. 7B) (SEQ ID NO:9)]; [S13Y, C17D, I69V, V84I, V91I,L98F, S118V, L122V, V146I, I157L, and T161A (see FIG. 7C) (SEQ IDNO:10)]; [S13F, C17D, V84I, V91I, L98F, S118A, L122I, I157L, and T161A(see FIG. 7D) (SEQ ID NO:11)]; [S13E, C17D, V84I, V91I, S118C, V146I,and T161C (see FIG. 8B) (SEQ ID NO:12)]; [S13A, V84I, V91I, S118C,V146I, I157L, and T161C (see FIG. 8C) (SEQ ID NO:13)]; [S13E, C17D,V84I, V91I, S118C, and T161C (see FIG. 8D) (SEQ ID NO:14)]; [S13E, C17D,I69V, V84I, V91I, S118A, L122I, V146I, I157L, and T161A (see FIG.9B)(SEQ ID NO:15)]; [S13E, C17D, V84I, V91I, S118A, V146I, and I157L(see FIG. 9C) (SEQ ID NO:16)]; and [S13E, C17D, V84I, V9I, S118A, L122I,I157L, and T161A (see FIG. 9D) (SEQ ID NO:17)].

Particularly preferred sequences for the B-chain of an IbA protein areselected from the group consisting of: [V84I and L87F (FIG. 10B) (SEQ IDNO:18)]; [A56L, L63F, V84I, L87F, V91I, and L122F (see FIG. 11B) (SEQ IDNO:19)]; [S13L, A56L, V84I, V91I, G114F, S118L, L122I, and (see FIG.12B) (SEQ ID NO:20)]; [S13L, C17A, A56L, V84I, L87F, V91L, G114F, S118L,L122I, and T161E (see FIG. 13B) (SEQ ID NO:21)]; [S13E, A56L, V84I,V91I, G114L, S118E, and T161E (see FIG. 14B) (SEQ ID NO:22)]; [C17T,A56L, V84I, V91I, G114L, S118E, and T161E (see FIG 15B) (SEQ ID NO:23)];and [C17T, A56L, V84I, V91I, S118E, and T161E (see FIG. 16B) (SEQ IDNO:24)].

In a preferred embodiment, the IbA proteins of the invention are humanIFN-β conformers. By “conformer” herein is meant a protein that has aprotein backbone 3D structure that is virtually the same but hassignificant differences in the amino acid side chains. That is, the IbAproteins of the invention define a conformer set, wherein all of theproteins of the set share a backbone structure and yet have sequencesthat differ by at least 3-5%. The three dimensional backbone structureof an IbA protein thus substantially corresponds to the threedimensional backbone structure of human IFN-β. “Backbone” in thiscontext means the non-side chain atoms: the nitrogen, carbonyl carbonand oxygen, and the α-carbon, and the hydrogens attached to the nitrogenand α-carbon. To be considered a conformer, a protein must have backboneatoms that are no more than 2 Å from the human IFN-β structure, with nomore than 1.5 Å being preferred, and no more than 1 Å being particularlypreferred. In general, these distances may be determined in two ways. Inone embodiment, each potential conformer is crystallized and its threedimensional structure determined. Alternatively, as the former is quitetedious, the sequence of each potential conformer is run in the PDAprogram to determine whether it is a conformer.

IbA proteins may also be identified as being encoded by IbA nucleicacids. In the case of the nucleic acid, the overall homology of thenucleic acid sequence is commensurate with amino acid homology but takesinto account the degeneracy in the genetic code and codon bias ofdifferent organisms. Accordingly, the nucleic acid sequence homology maybe either lower or higher than that of the protein sequence, with lowerhomology being preferred.

In a preferred embodiment, an IbA nucleic acid encodes an IbA protein.As will be appreciated by those in the art, due to the degeneracy of thegenetic code, an extremely large number of nucleic acids may be made,all of which encode the IbA proteins of the present invention. Thus,having identified a particular amino acid sequence, those skilled in theart could make any number of different nucleic acids, by simplymodifying the sequence of one or more codons in a way which does notchange the amino acid sequence of the IbA.

In one embodiment, the nucleic acid homology is determined throughhybridization studies. Thus, for example, nucleic acids which hybridizeunder high stringency to the nucleic acid sequence shown in FIG. 1 orits complement and encode a IbA protein is considered an IbA gene.

High stringency conditions are known in the art; see for exampleManiatis et al., Molecular Cloning: A Laboratory Manual, 2d Edition,1989, and Short Protocols in Molecular Biology, ed. Ausubel, et al.,both of which are hereby incorporated by reference. Stringent conditionsare sequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures. Anextensive guide to the hybridization of nucleic acids is found inTijssen, Techniques in Biochemistry and Molecular Biology—Hybridizationwith Nucleic Acid Probes, “Overview of principles of hybridization andthe strategy of nucleic acid assays” (1993). Generally, stringentconditions are selected to be about 5-10° C. lower than the thermalmelting point (T_(m)) for the specific sequence at a defined ionicstrength and pH. The T_(m) is the temperature (under defined ionicstrength, pH and nucleic acid concentration) at which 50% of the probescomplementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditionswill be those in which the salt concentration is less than about 1.0 Msodium ion, typically about 0.01 to 1.0 M sodium ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short probes (e.g. 10 to 50 nucleotides) and at least about 60°C. for long probes (e.g. greater than 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide.

In another embodiment, less stringent hybridization conditions are used;for example, moderate or low stringency conditions may be used, as areknown in the art; see Maniatis and Ausubel, supra, and Tijssen, supra.

The IbA proteins and nucleic acids of the present invention arerecombinant. As used herein, “nucleic acid” may refer to either DNA orRNA, or molecules which contain both deoxy- and ribonucleotides. Thenucleic acids include genomic DNA, cDNA and oligonucleotides includingsense and anti-sense nucleic acids. Such nucleic acids may also containmodifications in the ribose-phosphate backbone to increase stability andhalf life of such molecules in physiological environments.

The nucleic acid may be double stranded, single stranded, or containportions of both double stranded or single stranded sequence. As will beappreciated by those in the art, the depiction of a single strand(“Watson”) also defines the sequence of the other strand (“Crick”); thusthe sequence depicted in FIG. 1 also includes the complement of thesequence. By the term “recombinant nucleic acid” herein is meant nucleicacid, originally formed in vitro, in general, by the manipulation ofnucleic acid by endonucleases, in a form not normally found in nature.Thus an isolated IbA nucleic acid, in a linear form, or an expressionvector formed in vitro by ligating DNA molecules that are not normallyjoined, are both considered recombinant for the purposes of thisinvention. It is understood that once a recombinant nucleic acid is madeand reintroduced into a host cell or organism, it will replicatenon-recombinantly, i.e. using the in vivo cellular machinery of the hostcell rather than in vitro manipulations; however, such nucleic acids,once produced recombinantly, although subsequently replicatednon-recombinantly, are still considered recombinant for the purposes ofthe invention.

Similarly, a “recombinant protein” is a protein made using recombinanttechniques, i.e. through the expression of a recombinant nucleic acid asdepicted above. A recombinant protein is distinguished from naturallyoccurring protein by at least one or more characteristics. For example,the protein may be isolated or purified away from some or all of theproteins and compounds with which it is normally associated in its wildtype host, and thus may be substantially pure. For example, an isolatedprotein is unaccompanied by at least some of the material with which itis normally associated in its natural state, preferably constituting atleast about 0.5%, more preferably at least about 5% by weight of thetotal protein in a given sample. A substantially pure protein comprisesat least about 75% by weight of the total protein, with at least about80% being preferred, and at least about 90% being particularlypreferred. The definition includes the production of an IbA protein fromone organism in a different organism or host cell. Alternatively, theprotein may be made at a significantly higher concentration than isnormally seen, through the use of an inducible promoter or highexpression promoter, such that the protein is made at increasedconcentration levels. Furthermore, all of the IbA proteins outlinedherein are in a form not normally found in nature, as they contain aminoacid substitutions, insertions and deletions, with substitutions beingpreferred, as discussed below.

Also included within the definition of IbA proteins of the presentinvention are amino acid sequence variants of the IbA sequences outlinedherein and shown in the Figures. That is, the IbA proteins may containadditional variable positions as compared to human IFN-β. These variantsfall into one or more of three classes: substitutional, insertional ordeletional variants. These variants ordinarily are prepared by sitespecific mutagenesis of nucleotdes in the DNA encoding an IbA protein,using cassette or PCR mutagenesis or other techniques well known in theart, to produce DNA encoding the variant, and thereafter expressing theDNA in recombinant cell culture as outlined above. However, variant IbAprotein fragments having up to about 100-150 residues may be prepared byin vitro synthesis using established techniques. Amino acid sequencevariants are characterized by the predetermined nature of the variation,a feature that sets them apart from naturally occurring allelic orinterspecies variation of the IbA protein amino acid sequence. Thevariants typically exhibit the same qualitative biological activity asthe naturally occurring analogue, although variants can also be selectedwhich have modified characteristics as will be more fully outlinedbelow.

While the site or region for introducing an amino acid sequencevariation is predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed IbA variants screened for theoptimal combination of desired activity. Techniques for makingsubstitution mutations at predetermined sites in DNA having a knownsequence are well known, for example, M13 primer mutagenesis and PCRmutagenesis. Screening of the mutants is done using assays of IbAprotein activities.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about 1 to 20 amino acids, althoughconsiderably larger insertions may be tolerated. Deletions range fromabout 1 to about 20 residues, although in some cases deletions may bemuch larger.

Substitutions, deletions, insertions or any combination thereof may beused to arrive at a final derivative. Generally these changes are doneon a few amino acids to minimize the alteration of the molecule.However, larger changes may be tolerated in certain circumstances. Whensmall alterations in the characteristics of the IbA protein are desired,substitutions are generally made in accordance with the following chart:

CHART I Original Residue Exemplary Substitutions Ala Ser Arg Lys AsnGln, His Asp Glu Cys Ser, Ala Gln Asn Glu Asp Gly Pro His Asn, Gln IleLeu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, TyrSer Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those shown inChart I. For example, substitutions may be made which more significantlyaffect: the structure of the polypeptide backbone in the area of thealteration, for example the alpha-helical or beta-sheet structure; thecharge or hydrophobicity of the molecule at the target site; or the bulkof the side chain. The substitutions which in general are expected toproduce the greatest changes in the polypeptide's properties are thosein which (a) a hydrophilic residue, e.g. seryl or threonyl, issubstituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl,phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substitutedfor (or by) any other residue; (c) a residue having an electropositiveside chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by)an electronegative residue, e.g. glutamyl or aspartyl; or (d) a residuehaving a bulky side chain, e.g. phenylalanine, is substituted for (orby) one not having a side chain, e.g. glycine.

The variants typically exhibit the same qualitative biological activityand will elicit the same immune response as the original IbA protein,although variants also are selected to modify the characteristics of theIbA proteins as needed. Alternatively, the variant may be designed suchthat the biological activity of the IbA protein is altered. For example,glycosylation sites may be altered or removed. Similarly, the biologicalfunction may be altered; for example, in some instances it may bedesirable to have more or less potent IFN-β activity.

The IbA proteins and nucleic acids of the invention can be made in anumber of ways. Individual nucleic acids and proteins can be made asknown in the art and outlined below. Alternatively, libraries of IbAproteins can be made for testing.

In a preferred embodiment, sets or libraries of IbA proteins aregenerated from a probability distribution table. As outlined herein,there are a variety of methods of generating a probability distributiontable, including using PDA, sequence alignments, forcefield calculationssuch as SCMF calculations, etc. In addition, the probabilitydistribution can be used to generate information entropy scores for eachposition, as a measure of the mutational frequency observed in thelibrary.

In this embodiment, the frequency of each amino acid residue at eachvariable position in the list is identified. Frequencies can bethresholded, wherein any variant frequency lower than a cutoff is set tozero. This cutoff is preferably 1%, 2%, 5%, 10% or 20%, with 10% beingparticularly preferred. These frequencies are then built into the IbAlibrary. That is, as above, these variable positions are collected andall possible combinations are generated, but the amino acid residuesthat “fill” the library are utilized on a frequency basis. Thus, in anon-frequency based library, a variable position that has 5 possibleresidues will have 20% of the proteins comprising that variable positionwith the first possible residue, 20% with the second, etc. However, in afrequency based library, a variable position that has 5 possibleresidues with frequencies of 10%, 15%, 25%, 30% and 20%, respectively,will have 10% of the proteins comprising that variable position with thefirst possible residue, 15% of the proteins with the second residue, 25%with the third, etc. As will be appreciated by those in the art, theactual frequency may depend on the method used to actually generate theproteins; for example, exact frequencies may be possible when theproteins are synthesized. However, when the frequency-based primersystem outlined below is used, the actual frequencies at each positionwill vary, as outlined below.

As will be appreciated by those in the art and outlined herein,probability distribution tables can be generated in a variety of ways.In addition to the methods outlined herein, self-consistent mean field(SCMF) methods can be used in the direct generation of probabilitytables. SCMF is a deterministic computational method that uses a meanfield description of rotamer interactions to calculate energies. Aprobability table generated in this way can be used to create librariesas described herein. SCMF can be used in three ways: the frequencies ofamino acids and rotamers for each amino acid are listed at eachposition; the probabilities are determined directly from SCMF (seeDelarue et la. Pac. Symp. Biocomput. 109-21 (1997), expresslyincorporated by reference). In addition, highly variable positions andnon-variable positions can be identified. Alternatively, another methodis used to determine what sequence is jumped to during a search ofsequence space; SCMF is used to obtain an accurate energy for thatsequence; this energy is then used to rank it and create a rank-orderedlist of sequences (similar to a Monte Carlo sequence list). Aprobability table showing the frequencies of amino acids at eachposition can then be calculated from this list (Koehl et al., J. Mol.Biol. 239:249 (1994); Koehl et al., Nat. Struc. Biol. 2:163 (1995);Koehl et al., Curr. Opin. Struct. Biol. 6:222 (1996); Koehl et al., J.Mol. Bio. 293:1183 (1999); Koehl et al., J. Mol. Biol. 293:1161 (1999);Lee J. Mol. Biol. 236:918 (1994); and Vasquez Biopolymers 36:53-70(1995); all of which are expressly incorporated by reference. Similarmethods include, but are not limited to, OPLS-AA (Jorgensen, et al., J.Am. Chem. Soc. (1996), v 118, pp 11225-11236; Jorgensen, W. L.; BOSS,Version 4.1; Yale University: New Haven, Conn. (1999)); OPLS (Jorgensen,et al., J. Am. Chem. Soc. (1988), v 110, pp 1657ff; Jorgensen, et al., JAm. Chem. Soc. (1990), v 112, pp 4768ff); UNRES (United ResidueForcefield; Liwo, et al., Protein Science (1993), v 2, pp1697-1714;Liwo, et al., Protein Science (1993), v 2, pp1715-1731; Liwo, et al., J.Comp. Chem. (1997), v 18, pp849-873; Liwo, et al., J. Comp. Chem.(1997), v 18, pp874-884; Liwo, et al., J. Comp. Chem. (1998), v 19,pp259-276; Forcefield for Protein Structure Prediction (Liwo, et al.,Proc. Natl. Acad. Sci. USA (1999), v 96, pp5482-5485); ECEPP/3 (Liwo etal., J Protein Chem 1994 May;13(4):375-80); AMBER 1.1 force field(Weiner, et al., J. Am. Chem. Soc. v106, pp765-784); AMBER 3.0 forcefield (U.C. Singh et al., Proc. Natl. Acad. Sci. USA. 82:755-759);CHARMM and CHARMM22 (Brooks, et al., J. Comp. Chem. v4, pp 187-217);cvff3.0 (Dauber-Osguthorpe, et al., (1988) Proteins: Structure, Functionand Genetics, v4, pp31-47); cff91 (Maple, et al., J. Comp. Chem. v 5,162-182); also, the DISCOVER (cvff and cff9l) and AMBER forcefields areused in the INSIGHT molecular modeling package (Biosym/MSI, San DiegoCalif.) and HARMM is used in the QUANTA molecular modeling package(Biosym/MSI, San Diego Calif.).

In addition, as outlined herein, a preferred method of generating aprobability distribution table is through the use of sequence alignmentprograms. In addition, the probability table can be obtained by acombination of sequence alignments and computational approaches. Forexample, one can add amino acids found in the alignment of homologoussequences to the result of the computation. Preferable one can add thewild type amino acid identity to the probability table if it is notfound in the computation.

As will be appreciated, an IbA library created by recombining variablepositions and/or residues at the variable position may not be in arank-ordered list. In some embodiments, the entire list may just be madeand tested. Alternatively, in a preferred embodiment, the IbA library isalso in the form of a rank ordered list. This may be done for severalreasons, including the size of the library is still too big to generateexperimentally, or for predictive purposes. This may be done in severalways. In one embodiment, the library is ranked using the scoringfunctions of PDA to rank the library members. Alternatively, statisticalmethods could be used. For example, the library may be ranked byfrequency score; that is, proteins containing the most of high frequencyresidues could be ranked higher, etc. This may be done by adding ormultiplying the frequency at each variable position to generate anumerical score. Similarly, the library different positions could beweighted and then the proteins scored; for example, those containingcertain residues could be arbitrarily ranked.

In a preferred embodiment, the different protein members of the IbAlibrary may be chemically synthesized. This is particularly useful whenthe designed proteins are short, preferably less than 150 amino acids inlength, with less than 100 amino acids being preferred, and less than 50amino acids being particularly preferred, although as is known in theart, longer proteins can be made chemically or enzymatically. See forexample Wilken et al, Curr. Opin. Biotechnol. 9:412-26 (1998), herebyexpressly incorporated by reference.

In a preferred embodiment, particularly for longer proteins or proteinsfor which large samples are desired, the library sequences are used tocreate nucleic acids such as DNA which encode the member sequences andwhich can then be cloned into host cells, expressed and assayed, ifdesired. Thus, nucleic acids, and particularly DNA, can be made whichencodes each member protein sequence. This is done using well knownprocedures. The choice of codons, suitable expression vectors andsuitable host cells will vary depending on a number of factors, and canbe easily optimized as needed.

In a preferred embodiment, multiple PCR reactions with pooledoligonucleotides is done, as is generally depicted in FIG. 17. In thisembodiment, overlapping oligonucleotides are synthesized whichcorrespond to the full length gene. Again, these oligonucleotides mayrepresent all of the different amino acids at each variant position orsubsets.

In a preferred embodiment, these oligonucleotides are pooled in equalproportions and multiple PCR reactions are performed to create fulllength sequences containing the combinations of mutations defined by thelibrary. In addition, this may be done using error-prone PCR methods.

In a preferred embodiment, the different oligonucleotides are added inrelative amounts corresponding to the probability distribution table.The multiple PCR reactions thus result in full length sequences with thedesired combinations of mutations in the desired proportions.

The total number of oligonucleotides needed is a function of the numberof positions being mutated and the number of mutations being consideredat these positions: (number of oligos for constant positions)+M1+M2+M3+. . . Mn=(total number of oligos required), where Mn is the number ofmutations considered at position n in the sequence.

In a preferred embodiment, each overlapping oligonucleotide comprisesonly one position to be varied; in alternate embodiments, the variantpositions are too close together to allow this and multiple variants peroligonucleotide are used to allow complete recombination of all thepossibilities. That is, each oligo can contain the codon for a singleposition being mutated, or for more than one position being mutated. Themultiple positions being mutated must be close in sequence to preventthe oligo length from being impractical. For multiple mutating positionson an oligonucleotide, particular combinations of mutations can beincluded or excluded in the library by including or excluding theoligonucleotide encoding that combination. For example, as discussedherein, there may be correlations between variable regions; that is,when position X is a certain residue, position Y must (or must not) be aparticular residue. These sets of variable positions are sometimesreferred to herein as a “cluster”. When the clusters are comprised ofresidues close together, and thus can reside on one oligonucleotideprimer, the clusters can be set to the “good” correlations, andeliminate the bad combinations that may decrease the effectiveness ofthe library. However, if the residues of the cluster are far apart insequence, and thus will reside on different oligonucleotides forsynthesis, it may be desirable to either set the residues to the “good”correlation, or eliminate them as variable residues entirely. In analternative embodiment, the library may be generated in several steps,so that the cluster mutations only appear together. This procedure, i.e.the procedure of identifying mutation clusters and either placing themon the same oligonucleotides or eliminating them from the library orlibrary generation in several steps preserving clusters, canconsiderably enrich the experimental library with properly foldedprotein. Identification of clusters can be carried out by a number ofways, e.g. by using known pattern recognition methods, comparisons offrequencies of occurence of mutations or by using energy analysis of thesequences to be experimentally generated (for example, if the energy ofinteraction is high, the positions are correlated). These correlationsmay be positional correlations (e.g. variable positions 1 and 2 alwayschange together or never change together) or sequence correlations (e.g.if there is residue A at position 1, there is always residue B atposition 2). See: Pattern discovery in Biomolecular Data: Tools,Techniques, and Applications; edited by Jason T. L. Wang, Bruce A.Shapiro, Dennis Shasha. New York: Oxford University, 1999; Andrews,Harry C. Introduction to mathematical techniques in pattern recognition;New York, Wiley-lnterscience [1 972]; Applications of PatternRecognition; Editor, K. S. Fu. Boca Raton, Fla. CRC Press, 1982; GeneticAlgorithms for Pattern Recognition; edited by Sankar K. Pal, Paul P.Wang. Boca Raton: CRC Press, c1996; Pandya, Abhijit S., Patternrecognition with neural networks in C++/Abhijit S. Pandya, Robert B.Macy. Boca Raton, Fla.: CRC Press, 1996; Handbook of pattern recognition& computer vision I edited by C. H. Chen, L. F. Pau, P. S. P. Wang. 2nded. Singapore; River Edge, N.J.: World Scientific, c1999; Friedman,Introduction to Pattern Recognition: Statistical, Structural, Neural,and Fuzy Logic Approaches; River Edge, N.J.: World Scientific, c1999,Series title: Series in machine perception and artificial intelligence;vol. 32; all of which are expressly incorporated by reference. Inaddition, programs used to search for consensus motifs can be used aswell.

In addition, correlations and shuffling can be fixed or optimized byaltering the design of the oligonucleotides; that is, by deciding wherethe oligonucleotides (primers) start and stop (e.g. where the sequencesare “cut”). The start and stop sites of oligos can be set to maximizethe number of clusters that appear in single oligonucleotides, therebyenriching the library with higher scoring sequences. Differentoligonucleotide start and stop site options can be computationallymodeled and ranked according to number of clusters that are representedon single oligos, or the percentage of the resulting sequencesconsistent with the predicted library of sequences.

The total number of oligonucleotides required increases when multiplemutable positions are encoded by a single oligonucleotide. The annealedregions are the ones that remain constant, i.e. have the sequence of thereference sequence.

Oligonucleotides with insertions or deletions of codons can be used tocreate a library expressing different length proteins. In particularcomputational sequence screening for insertions or deletions can resultin secondary libraries defining different length proteins, which can beexpressed by a library of pooled oligonucleotide of different lengths.

In a preferred embodiment, the IbA library is done by shuffling thefamily (e.g. a set of variants); that is, some set of the top sequences(if a rank-ordered list is used) can be shuffled, either with or withouterror-prone PCR. “Shuffling” in this context means a recombination ofrelated sequences, generally in a random way. It can include “shuffling”as defined and exemplified in U.S. Pat. Nos. 5,830,721; 5,811,238;5,605,793; 5,837,458 and PCT US/19256, all of which are expresslyincorporated by reference in their entirety. This set of sequences canalso be an artificial set; for example, from a probability table (forexample generated using SCMF) or a Monte Carlo set. Similarly, the“family” can be the top 10 and the bottom 10 sequences, the top 100sequence, etc. This may also be done using error-prone PCR.

Thus, in a preferred embodiment, in silico shuffling is done using thecomputational methods described herein. That is, starting with eithertwo libraries or two sequences, random recombinations of the sequencescan be generated and evaluated.

In a preferred embodiment, error-prone PCR is done to generate the IbAlibrary. See U.S. Pat. Nos. 5,605,793, 5,811,238, and 5,830,721, all ofwhich are hereby incorporated by reference. This can be done on theoptimal sequence or on top members of the library, or some otherartificial set or family. In this embodiment, the gene for the optimalsequence found in the computational screen of the primary library can besynthesized. Error prone PCR is then performed on the optimal sequencegene in the presence of oligonucleofides that code for the mutations atthe variant positions of the library (bias oligonucleotides). Theaddition of the oligonucleotdes will create a bias favoring theincorporation of the mutations in the library. Alternatively, onlyoligonucleotdes for certain mutations may be used to bias the library.

In a preferred embodiment, gene shuffling with error prone PCR can beperformed on the gene for the optimal sequence, in the presence of biasoligonucleotides, to create a DNA sequence library that reflects theproportion of the mutations found in the IbA library. The choice of thebias oligonucleotides can be done in a variety of ways; they can bechosen on the basis of their frequency, i.e. oligonucleotides encodinghigh mutational frequency positions can be used; alternatively,oligonucleotides containing the most variable positions can be used,such that the diversity is increased; if the secondary library isranked, some number of top scoring positions can be used to generatebias oligonucleotides; random positions may be chosen; a few top scoringand a few low scoring ones may be chosen; etc. What is important is togenerate new sequences based on preferred variable positions andsequences.

In a preferred embodiment, PCR using a wild type gene or other gene canbe used, as is schematically depicted in FIG. 18. In this embodiment, astarting gene is used; generally, although this is not required, thegene is usually the wild type gene. In some cases it may be the geneencoding the global optimized sequence, or any other sequence of thelist, or a consensus sequence obtained e.g. from aligning homologoussequences from different organisms. In this embodiment, oligonucleotidesare used that correspond to the variant positions and contain thedifferent amino acids of the library. PCR is done using PCR primers atthe termini, as is known in the art. This provides two benefits; thefirst is that this generally requires fewer oligonucleotides and canresult in fewer errors. In addition, it has experimental advantages inthat if the wild type gene is used, it need not be synthesized.

In addition, there are several other techniques that can be used, asexemplified in the figures, e.g. FIGS. 19-21. In a preferred embodiment,ligation of PCR products is done.

In a preferred embodiment, a variety of additional steps may be done tothe IbA library; for example, further computational processing canoccur, different IbA libraries can be recombined, or cutoffs fromdifferent libraries can be combined. In a preferred embodiment, an IbAlibrary may be computationally remanipulated to form an additional IbAlibrary (sometimes referred to herein as “tertiary libraries”). Forexample, any of the IbA library sequences may be chosen for a secondround of PDA, by freezing or fixing some or all of the changed positionsin the first library. Alternatively, only changes seen in the lastprobability distribution table are allowed. Alternatively, thestringency of the probability table may be altered, either by increasingor decreasing the cutoff for inclusion. Similarly, the IbA library maybe recombined experimentally after the first round; for example, thebest gene/genes from the first screen may be taken and gene assemblyredone (using techniques outlined below, multiple PCR, error prone PCR,shuffling, etc.). Alternatively, the fragments from one or more goodgene(s) to change probabilities at some positions. This biases thesearch to an area of sequence space found in the first round ofcomputational and experimental screening.

In a preferred embodiment, a tertiary library can be generated fromcombining different IbA libraries. For example, a probabilitydistribution table from a first IbA library can be generated andrecombined, either computationally or experimentally, as outlinedherein. A PDA IbA library may be combined with a sequence alignment IbAlibrary, and either recombined (again, computationally orexperimentally) or just the cutoffs from each joined to make a newtertiary library. The top sequences from several libraries can berecombined. Sequences from the top of a library can be combined withsequences from the bottom of the library to more broadly sample sequencespace, or only sequences distant from the top of the library can becombined. IbA libraries that analyzed different parts of a protein canbe combined to a tertiary library that treats the combined parts of theprotein.

In a preferred embodiment, a tertiary library can be generated usingcorrelations in an IbA library. That is, a residue at a first variableposition may be correlated to a residue at second variable position (orcorrelated to residues at additional positions as well). For example,two variable positions may sterically or electrostatically interact,such that if the first residue is X, the second residue must be Y. Thismay be either a positive or negative correlation.

Using the nucleic acids of the present invention which encode an IbAprotein, a variety of expression vectors are made. The expressionvectors may be either self-replicating extrachromosomal vectors orvectors which integrate into a host genome. Generally, these expressionvectors include transcriptional and translational regulatory nucleicacid operably linked to the nucleic acid encoding the IbA protein. Theterm “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation.

In a preferred embodiment, when the endogenous secretory sequence leadsto a low level of secretion of the naturally occurring protein or of theIbA protein, a replacement of the naturally occurring secretory leadersequence is desired. In this embodiment, an unrelated secretory leadersequence is operably linked to an IbA encoding nucleic acid leading toincreased protein secretion. Thus, any secretory leader sequenceresulting in enhanced secretion of the IbA protein, when compared to thesecretion of IFN-β and its secretory sequence, is desired. Suitablesecretory leader sequences that lead to the secretion of a protein areknow in the art.

In another preferred embodiment, a secretory leader sequence of anaturally occurring protein or a protein is removed by techniques knownin the art and subsequent expression results in intracellularaccumulation of the recombinant protein.

Generally, “operably linked” means that the DNA sequences being linkedare contiguous, and, in the case of a secretory leader, contiguous andin reading phase. However, enhancers do not have to be contiguous.Linking is accomplished by ligation at convenient restriction sites. Ifsuch sites do not exist, the synthetic oligonucleotide adaptors orlinkers are used in accordance with conventional practice. Thetranscriptional and translational regulatory nucleic acid will generallybe appropriate to the host cell used to express the fusion protein; forexample, transcriptional and translational regulatory nucleic acidsequences from Bacillus are preferably used to express the fusionprotein in Bacillus. Numerous types of appropriate expression vectors,and suitable regulatory sequences are known in the art for a variety ofhost cells.

In general, the transcriptional and translational regulatory sequencesmay include, but are not limited to, promoter sequences, ribosomalbinding sites, transcriptional start and stop sequences, translationalstart and stop sequences, and enhancer or activator sequences. In apreferred embodiment, the regulatory sequences include a promoter andtranscriptional start and stop sequences.

Promoter sequences encode either constitutive or inducible promoters.The promoters may be either naturally occurring promoters or hybridpromoters. Hybrid promoters, which combine elements of more than onepromoter, are also known in the art, and are useful in the presentinvention. In a preferred embodiment, the promoters are strongpromoters, allowing high expression in cells, particularly mammaliancells, such as the CMV promoter, particularly in combination with a Tetregulatory element.

In addition, the expression vector may comprise additional elements. Forexample, the expression vector may have two replication systems, thusallowing it to be maintained in two organisms, for example in mammalianor insect cells for expression and in a prokaryotic host for cloning andamplification. Furthermore, for integrating expression vectors, theexpression vector contains at least one sequence homologous to the hostcell genome, and preferably two homologous sequences which flank theexpression construct. The integrating vector may be directed to aspecific locus in the host cell by selecting the appropriate homologoussequence for inclusion in the vector. Constructs for integrating vectorsare well known in the art.

In addition, in a preferred embodiment, the expression vector contains aselectable marker gene to allow the selection of transformed host cells.Selection genes are well known in the art and will vary with the hostcell used.

A preferred expression vector system is a retroviral vector system suchas is generally described in PCT/US97/01019 and PCT/US97/01048, both ofwhich are hereby expressly incorporated by reference.

In a preferred embodiment, the expression vector comprises thecomponents described above and a gene encoding an IbA protein. In thisaspect, only one species of an IbA protein will be expressed in the cellcomprising the expression vector. In one aspect of this embodiment, itis desired to express an optimized A-chain of IFN-β and an optimizedB-chain of IFN-β within the same cell and thus, two expression vectors,one comprising a gene coding for an optimized A-chain of IFN-β, theother one comprising a gene coding for an optimized B-chain of IFN-β areintroduced into the same host cell. This allows formation of a preferredIbA dimer.

In another aspect of this embodiment, an expression vector isconstructed that comprises two IbA genes encoding two different IbAproteins. In this embodiment, one IbA gene encodes an optimized A chainof IFN-β and the second gene encodes an optimized B-chain of IFN-β. Inone aspect of this embodiment, a polycistronic gene can be constructedas is known in the art for co-expression in a host cell.

As will be appreciated by those in the art, all combinations arepossible and accordingly, as used herein, the combination of components,comprised by one or more vectors, which may be retroviral or not, isreferred to herein as a “vector composition”.

The IbA nucleic acids are introduced into the cells either alone or incombination with an expression vector. By “introduced into” orgrammatical equivalents herein is meant that the nucleic acids enter thecells in a manner suitable for subsequent expression of the nucleicacid. The method of introduction is largely dictated by the targetedcell type, discussed below. Exemplary methods include (Ca₃PO₄)₂precipitation, liposome fusion, lipofectin®, electroporation, viralinfection, etc. The IbA nucleic acids may stably integrate into thegenome of the host cell (for example, with retroviral introduction,outlined below), or may exist either transiently or stably in thecytoplasm (i.e. through the use of traditional plasmids, utilizingstandard regulatory sequences, selection markers, etc.).

The IbA proteins of the present invention are produced by culturing ahost cell transformed with an expression vector containing nucleic acidencoding an IbA A protein, under the appropriate conditions to induce orcause expression of the IbA protein. The conditions appropriate for IbAprotein expression will vary with the choice of the expression vectorand the host cell, and will be easily ascertained by one skilled in theart through routine experimentation. For example, the use ofconstitutive promoters in the expression vector will require optimizingthe growth and proliferation of the host cell, while the use of aninducible promoter requires the appropriate growth conditions forinduction. In addition, in some embodiments, the timing of the harvestis important. For example, the baculoviral systems used in insect cellexpression are lytic viruses, and thus harvest time selection can becrucial for product yield.

Appropriate host cells include yeast, bacteria, archebacteria, fungi,and insect and animal cells, including mammalian cells. Of particularinterest are Drosophila melangaster cells, Saccharomyces cerevisiae andother yeasts, E. coli, Bacillus subtilis, SF9 cells, C129 cells, 293cells, Neurospora, BHK, CHO, COS, Pichia Pastoris, etc.

In a preferred embodiment, the IbA proteins are expressed in mammaliancells. Mammalian expression systems are also known in the art, andinclude retroviral systems. A mammalian promoter is any DNA sequencecapable of binding mammalian RNA polymerase and initiating thedownstream (3′) transcription of a coding sequence for the fusionprotein into mRNA. A promoter will have a transcription initiatingregion, which is usually placed proximal to the 5′ end of the codingsequence, and a TATA box, using a located 25-30 base pairs upstream ofthe transcription initiation site. The TATA box is thought to direct RNApolymerase II to begin RNA synthesis at the correct site. A mammalianpromoter will also contain an upstream promoter element (enhancerelement), typically located within 100 to 200 base pairs upstream of theTATA box. An upstream promoter element determines the rate at whichtranscription is initiated and can act in either orientation. Ofparticular use as mammalian promoters are the promoters from mammalianviral genes, since the viral genes are often highly expressed and have abroad host range. Examples include the SV40 early promoter, mousemammary tumor virus LTR promoter, adenovirus major late promoter, herpessimplex virus promoter, and the CMV promoter.

Typically, transcription termination and polyadenylation sequencesrecognized by mammalian cells are regulatory regions located 3′ to thetranslation stop codon and thus, together with the promoter elements,flank the coding sequence. The 3′ terminus of the mature mRNA is formedby site-specific post-translational cleavage and polyadenylation.Examples of transcription terminator and polyadenlytion signals includethose derived form SV40.

The methods of introducing exogenous nucleic acid into mammalian hosts,as well as other hosts, is well known in the art, and will vary with thehost cell used. Techniques include dextran-mediated transfection,calcium phosphate precipitation, polybrene mediated transfection,protoplast fusion, electroporation, viral infection, encapsulation ofthe polynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei. As outlined herein, a particularly preferred methodutilizes retroviral infection, as outlined in PCT US97/01019,incorporated by reference.

As will be appreciated by those in the art, the type of mammalian cellsused in the present invention can vary widely. Basically, any mammaliancells may be used, with mouse, rat, primate and human cells beingparticularly preferred, although as will be appreciated by those in theart, modifications of the system by pseudotyping allows all eukaryoticcells to be used, preferably higher eukaryotes. As is more fullydescribed below, a screen will be set up such that the cells exhibit aselectable phenotype in the presence of a bioactive peptide. As is morefully described below, cell types implicated in a wide variety ofdisease conditions are particularly useful, so long as a suitable screenmay be designed to allow the selection of cells that exhibit an alteredphenotype as a consequence of the presence of a peptide within the cell.

Accordingly, suitable cell types include, but are not limited to, tumorcells of all types (particularly melanoma, myeloid leukemia, carcinomasof the lung, breast, ovaries, colon, kidney, prostate, pancreas andtestes), cardiomyocytes, endothelial cells, epithelial cells,lymphocytes (T-cell and B cell) , mast cells, eosinophils, vascularintimal cells, hepatocytes, leukocytes including mononuclear leukocytes,stem cells such as haemopoetic, neural, skin, lung, kidney, liver andmyocyte stem cells (for use in screening for differentiation andde-differentiation factors), osteoclasts, chondrocytes and otherconnective tissue cells, keratinocytes, melanocytes, liver cells, kidneycells, and adipocytes. Suitable cells also include known research cells,including, but not limited to, Jurkat T cells, NIH3T3 cells, CHO, Cos,etc. See the ATCC cell line catalog, hereby expressly incorporated byreference.

In one embodiment, the cells may be additionally genetically engineered,that is, contain exogeneous nucleic acid other than the IbA nucleicacid.

In a preferred embodiment, the IbA proteins are expressed in bacterialsystems. Bacterial expression systems are well known in the art.

A suitable bacterial promoter is any nucleic acid sequence capable ofbinding bacterial RNA polymerase and initiating the downstream (3′)transcription of the coding sequence of the IbA protein into mRNA. Abacterial promoter has a transcription initiation region which isusually placed proximal to the 5′ end of the coding sequence. Thistranscription initiation region typically includes an RNA polymerasebinding site and a transcription initiation site. Sequences encodingmetabolic pathway enzymes provide particularly useful promotersequences. Examples include promoter sequences derived from sugarmetabolizing enzymes, such as galactose, lactose and maltose, andsequences derived from biosynthetic enzymes such as tryptophan.Promoters from bacteriophage may also be used and are known in the art.In addition, synthetic promoters and hybrid promoters are also useful;for example, the tac promoter is a hybrid of the trp and lac promotersequences. Furthermore, a bacterial promoter can include naturallyoccurring promoters of non-bacterial origin that have the ability tobind bacterial RNA polymerase and initiate transcription.

In addition to a functioning promoter sequence, an efficient ribosomebinding site is desirable. In E. coli, the ribosome binding site iscalled the Shine-Delgarno (SD) sequence and includes an initiation codonand a sequence 3-9 nucleotides in length located 3-11 nucleotidesupstream of the initiation codon.

The expression vector may also include a signal peptide sequence thatprovides for secretion of the IbA protein in bacteria. The signalsequence typically encodes a signal peptide comprised of hydrophobicamino acids which direct the secretion of the protein from the cell, asis well known in the art. The protein is either secreted into the growthmedia (gram-positive bacteria) or into the periplasmic space, locatedbetween the inner and outer membrane of the cell (gram-negativebacteria). For expression in bacteria, usually bacterial secretoryleader sequences, operably linked to an IbA encoding nucleic acid, arepreferred.

The bacterial expression vector may also include a selectable markergene to allow for the selection of bacterial strains that have beentransformed. Suitable selection genes include genes which render thebacteria resistant to drugs such as ampicillin, chloramphenicol,erythromycin, kanamycin, neomycin and tetracycline. Selectable markersalso include biosynthetic genes, such as those in the histidine,tryptophan and leucine biosynthetic pathways.

These components are assembled into expression vectors. Expressionvectors for bacteria are well known in the art, and include vectors forBacillus subtilis, E. coli, Streptococcus cremoris, and Streptococcuslividans, among others.

The bacterial expression vectors are transformed into bacterial hostcells using techniques well known in the art, such as calcium chloridetreatment, electroporation, and others.

In one embodiment, IbA proteins are produced in insect cells. Expressionvectors for the transformation of insect cells, and in particular,baculovirus-based expression vectors, are well known in the art.

In a preferred embodiment, IbA protein is produced in yeast cells. Yeastexpression systems are well known in the art, and include expressionvectors for Saccharomyces cerevisiae, Candida albicans and C. maltosa,Hansenula polymorpha, Kluyveromyces fragilis and K. lactis, Pichiaguillerimondii and P. pastoris, Schizosaccharomyces pombe, and Yarrowialipolytica. Preferred promoter sequences for expression in yeast includethe inducible GAL1,10 promoter, the promoters from alcoholdehydrogenase, enolase, glucokinase, glucose-6-phosphate isomerase,glyceraldehyde-3-phosphate-dehydrogenase, hexokinase,phosphofructokinase, 3-phosphoglycerate mutase, pyruvate kinase, and theacid phosphatase gene. Yeast selectable markers include ADE2, HIS4,LEU2, TRP1, and ALG7, which confers resistance to tunicamycin; theneomycin phosphotransferase gene, which confers resistance to G418; andthe CUP1 gene, which allows yeast to grow in the presence of copperions.

In addition, the IbA polypeptides of the invention may be further fusedto other proteins, if desired, for example to increase expression orstabilize the protein.

In one embodiment, the IbA nucleic acids, proteins and antibodies of theinvention are labeled with a label other than the scaffold. By “labeled”herein is meant that a compound has at least one element, isotope orchemical compound attached to enable the detection of the compound. Ingeneral, labels fall into three classes: a) isotopic labels, which maybe radioactive or heavy isotopes; b) immune labels, which may beantibodies or antigens; and c) colored or fluorescent dyes. The labelsmay be incorporated into the compound at any position.

Once made, the IbA proteins may be covalently modified. One type ofcovalent modification includes reacting targeted amino acid residues ofan IbA polypeptide with an organic derivatizing agent that is capable ofreacting with selected side chains or the N-or C-terminal residues of anIbA polypeptide. Derivatization with bifunctional agents is useful, forinstance, for crosslinking an IbA protein to a water-insoluble supportmatrix or surface for use in the method for purifying anti-IbAantibodies or screening assays, as is more fully described below.Commonly used crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of the“-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylaffon of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the IbA polypepfide includedwithin the scope of this invention comprises altering the nativeglycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence IbApolypeptide, and/or adding one or more glycosylation sites that are notpresent in the native sequence IbA polypeptide.

Addition of glycosylation sites to IbA polypeptides may be accomplishedby altering the amino acid sequence thereof. The alteration may be made,for example, by the addition of, or substitution by, one or more serineor threonine residues to the native sequence IbA polypeptide (forO-linked glycosylation sites). The IbA amino acid sequence mayoptionally be altered through changes at the DNA level, particularly bymutating the DNA encoding the IbA polypeptide at preselected bases suchthat codons are generated that will translate into the desired aminoacids.

Another means of increasing the number of carbohydrate moieties on theIbA polypeptide is by chemical or enzymatic coupling of glycosides tothe polypeptide. Such methods are described in the art, e.g., inWO87/05330 published Sep. 11, 1987, and in Aplin and Wriston, CRC Crit.Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the IbA polypeptide may beaccomplished chemically or enzymatically or by mutational substitutionof codons encoding for amino acid residues that serve as targets forglycosylabon. Chemical deglycosylation techniques are known in the artand described, for instance, by Hakimuddin, et al., Arch. Biochem.Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131(1981). Enzymatic cleavage of carbohydrate moieties on polypeptides canbe achieved by the use of a variety of endo-and exo-glycosidases asdescribed by Thotakura et al., Meth. Enzymol., 138:350 (1987).

Such derivatized moieties may improve the solubility, absorption,permeability across the blood brain barrier, biological half life, andthe like. Such moieties or modifications of IbA polypeptides mayalternatively eliminate or attenuate any possible undesirable sideeffect of the protein and the like. Moieties capable of mediating sucheffects are disclosed, for example, in Remington's PharmaceuticalSciences, 16th ed., Mack Publishing Co., Easton, Pa. (1980).

Another type of covalent modification of IbA comprises linking the IbApolypeptide to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in themanner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

IbA polypeptides of the present invention may also be modified in a wayto form chimeric molecules comprising an IbA polypeptide fused toanother, heterologous polypeptide or amino acid sequence. In oneembodiment, such a chimeric molecule comprises a fusion of an IbApolypeptide with a tag polypeptide which provides an epitope to which ananti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino-or carboxyl-terminus of the IbA polypeptide. Thepresence of such epitope-tagged forms of an IbA polypeptide can bedetected using an antibody against the tag polypeptide. Also, provisionof the epitope tag enables the IbA polypepbde to be readily purified byaffinity purification using an anti-tag antibody or another type ofaffinity matrix that binds to the epitope tag. In an alternativeembodiment, the chimeric molecule may comprise a fusion of an IbApolypeptide with an immunoglobulin or a particular region of animmunoglobulin. For a bivalent form of the chimeric molecule, such afusion could be to the Fc region of an IgG molecule.

Various tag polypeptides and their respective antibodies are well knownin the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol. 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptdes include the Flag-peptide [Hopp et al.,BioTechnology 6:1204-1210 (1988)]; the KT3 epitope peptde [Martin etal., Science 255:192-1944 (1992)]; tubulin epitope peptide [Skinner etal., J. Biol. Chem. 266:15163-15166 (1991)]; and the T7 gene 10 proteinpeptde tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. U.S.A.87:6393-6397 (1990)].

In a preferred embodiment, the IbA protein is purified or isolated afterexpression. IbA proteins may be isolated or purified in a variety ofways known to those skilled in the art depending on what othercomponents are present in the sample. Standard purification methodsinclude electrophoretic, molecular, immunological and chromatographictechniques, including ion exchange, hydrophobic, affinity, andreverse-phase HPLC chromatography, and chromatofocusing. For example,the IbA protein may be purified using a standard anti-library antibodycolumn. Ultrafiltration and diafiltration techniques, in conjunctionwith protein concentration, are also useful. For general guidance insuitable purification techniques, see Scopes, R., Protein Purification,Springer-Verlag, NY (1982). The degree of purification necessary willvary depending on the use of the IbA protein. In some instances nopurification will be necessary.

Once made, the IbA proteins and nucleic acids of the invention find usein a number of applications. In a preferred embodiment, the IbA proteinsare administered to a patent to treat an IFN-β-associated disorder.

By “IFN-β associated disorder” or “IFN-β responsive disorder” or“condition” herein is meant a disorder that can be ameliorated by theadministration of a pharamaceutical composition comprising an IFN-β orIbA protein, including, but not limited to, multiple sclerosis;idiopathic pulmonary fibrosis; inflammatory diseases; viral diseases;infections caused by papilloma viruses, such as genital warts andcondylomata of the uterine cervix; infections caused by hepatitisviruses, such as acute/chronic hepatitis B and non-A, non-B hepatitis(hepatitis C); infections caused by herpes viruses, such as herpesgenitalis, herpes zoster, herpes keratitis, and herpes simplex; viralencephalitis; cytomegalovirus pneumonia; prophylaxis of rhinovirus;cancer, including several malignant diseases such as osteosarcoma, basalcell carcinoma, cervical dysplasia, glioma, acute myeloid leukemia,multiple myeloma, Hodgkin's disease, melanoma, renal cancer, livercancer, and breast cancer.

In a preferred embodiment, a therapeutically effective dose of an IbAprotein is administered to a patient in need of treatment. By“therapeutically effective dose” herein is meant a dose that producesthe effects for which it is administered. The exact dose will depend onthe purpose of the treatment, and will be ascertainable by one skilledin the art using known techniques. In a preferred embodiment, dosages ofabout 5 μg/kg are used, administered either intraveneously orsubcutaneously. As is known in the art, adjustments for IbA proteindegradation, systemic versus localized delivery, and rate of newprotease synthesis, as well as the age, body weight, general health,sex, diet, time of administration, drug interaction and the severity ofthe condition may be necessary, and will be ascertainable with routineexperimentation by those skilled in the art.

A “patient” for the purposes of the present invention includes bothhumans and other animals, particularly mammals, and organisms. Thus themethods are applicable to both human therapy and veterinaryapplications. In the preferred embodiment the patient is a mammal, andin the most preferred embodiment the patient is human.

The term “treatment” in the instant invention is meant to includetherapeutic treatment, as well as prophylactic, or suppressive measuresfor the disease or disorder. Thus, for example, in the case of multiplesclerosis, successful administration of an IbA protein prior to onset ofthe disease results in “treatment” of the disease. As another example,successful administration of an IbA protein after clinical manifestationof the disease to combat the symptoms of the disease comprisestreatment” of the disease. “Treatment” also encompasses administrationof an IbA protein after the appearance of the disease in order toeradicate the disease. Successful administration of an agent after onsetand after clinical symptoms have developed, with possible abatement ofclinical symptoms and perhaps amelioration of the disease, comprises“treatment” of the disease.

Those “in need of treatment” include mammals, in particular humans,already having the disease or disorder, as well as those prone to havingthe disease or disorder, including those in which the disease ordisorder is to be prevented.

In another embodiment, a therapeutically effective dose of an IbAprotein, an IbA gene, or an IbA antibody is administered to a patienthaving a disease involving inappropriate expression of IFN-β. A “diseaseinvolving inappropriate expression of a IFN-β” within the scope of thepresent invention is meant to include diseases or disorderscharacterized by an overabundance of IFN-β. This overabundance may bedue to any cause, including, but not limited to, overexpression at themolecular level, prolonged or accumulated appearance at the site ofaction, or increased activity of IFN-β relative to normal. Includedwithin this definition are diseases or disorders characterized by areduction of IFN-β. This reduction may be due to any cause, including,but not limited to, reduced expression at the molecular level, shortenedor reduced appearance at the site of action, or decreased activity ofIFN-β relative to normal. Such an overabundance or reduction of IFN-βcan be measured relative to normal expression, appearance, or activityof IFN-β according to, but not limited to, the assays described andreferenced herein.

The administration of the IbA proteins of the present invention,preferably in the form of a sterile aqueous solution, can be done in avariety of ways, including, but not limited to, orally, subcutaneously,intravenously, intranasally, transdermally, intraperitoneally,intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly.In some instances, for example, in the treatment of wounds,inflammation, or multiple sclerosis, the IbA A protein may be directlyapplied as a solution or spray. Depending upon the manner ofintroduction, the pharmaceutical composition may be formulated in avariety of ways. The concentration of the therapeutically active IbAprotein in the formulation may vary from about 0.1 to 100 weight %. Inanother preferred embodiment, the concentration of the IbA protein is inthe range of 0.003 to 1.0 molar, with dosages from 0.03, 0.05, 0.1, 0.2,and 0.3 millimoles per kilogram of body weight being preferred.

The pharmaceutical compositions of the present invention comprise an IbAprotein in a form suitable for administration to a patient. In thepreferred embodiment, the pharmaceutical compositions are in a watersoluble form, such as being present as pharmaceutically acceptablesalts, which is meant to include both acid and base addition salts.“Pharmaceutically acceptable acid addition salt” refers to those saltsthat retain the biological effectiveness of the free bases and that arenot biologically or otherwise undesirable, formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as acetic acid,propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid,malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and thelike. “Pharmaceutically acceptable base addition salts” include thosederived from inorganic bases such as sodium, potassium, lithium,ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminumsalts and the like. Particularly preferred are the ammonium, potassium,sodium, calcium, and magnesium salts. Salts derived frompharmaceutically acceptable organic non-toxic bases include salts ofprimary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, and ethanolamine.

The pharmaceutical compositions may also include one or more of thefollowing: carrier proteins such as serum albumin; buffers such asNaOAc; fillers such as microcrystalline cellulose, lactose, corn andother starches; binding agents; sweeteners and other flavoring agents;coloring agents; and polyethylene glycol. Additives are well known inthe art, and are used in a variety of formulations.

In addition, in one embodiment, the IbA proteins of the presentinvention are formulated using a process for pharmaceutical compositionsof recombinant IFN-β as described in U.S. Pat. No. 5,183,746 which,hereby, is expressly incorporated in its entirety.

In a further embodiment, the IbA proteins are added in a micellularformulation; see U.S. Pat. No. 5,833,948, hereby expressly incorporatedby reference in its entirety.

Combinations of pharmaceutical compositions may be administered.Moreover, the compositions may be administered in combination with othertherapeutics.

In one embodiment provided herein, antibodies, including but not limitedto monoclonal and polyclonal antibodies, are raised against IbA proteinsusing methods known in the art. In a preferred embodiment, these ant-IbAantibodies are used for immunotherapy. Thus, methods of immunotherapyare provided. By “immunotherapy” is meant treatment of an IFN-β relateddisorders with an antibody raised against an IbA protein. As usedherein, immunotherapy can be passive or active. Passive immunotherapy,as defined herein, is the passive transfer of antibody to a recipient(patient). Active immunization is the induction of antibody and/orT-cell responses in a recipient (patient). Induction of an immuneresponse can be the consequence of providing the recipient with an IbAprotein antigen to which antibodies are raised. As appreciated by one ofordinary skill in the art, the IbA protein antigen may be provided byinjecting an IbA polypeptide against which antibodies are desired to beraised into a recipient, or contacting the recipient with an IbA proteinencoding nucleic acid, capable of expressing the IbA protein antigen,under conditions for expression of the IbA protein antigen.

In another preferred embodiment, a therapeutic compound is conjugated toan antibody, preferably an ant-IbA protein antibody. The therapeuticcompound may be a cytotoxic agent. In this method, targeting thecytotoxic agent to tumor tissue or cells, results in a reduction in thenumber of afflicted cells, thereby reducing symptoms associated withcancer, and IbA protein related disorders. Cytotoxic agents are numerousand varied and include, but are not limited to, cytotoxic drugs ortoxins or active fragments of such toxins. Suitable toxins and theircorresponding fragments include diptheria A chain, exotoxin A chain,ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin andthe like. Cytotoxic agents also include radiochemicals made byconjugating radioisotopes to antibodies raised against cell cycleproteins, or binding of a radionuclide to a chelating agent that hasbeen covalently attached to the antibody.

In a preferred embodiment, IbA proteins are administered as therapeuticagents, and can be formulated as outlined above. Similarly, IbA genes(including both the full-length sequence, partial sequences, orregulatory sequences of the IbA coding regions) can be administered ingene therapy applications, as is known in the art. These IbA genes caninclude antisense applications, either as gene therapy (i.e. forincorporation into the genome) or as antisense compositions, as will beappreciated by those in the art.

In a preferred embodiment, the nucleic acid encoding the IbA proteinsmay also be used in gene therapy. In gene therapy applications, genesare introduced into cells in order to achieve in vivo synthesis of atherapeutically effective genetic product, for example for replacementof a defective gene. “Gene therapy” includes both conventional genetherapy where a lasting effect is achieved by a single treatment, andthe administration of gene therapeutic agents, which involves the onetime or repeated administration of a therapeutically effective DNA ormRNA. Antisense RNAs and DNAs can be used as therapeutic agents forblocking the expression of certain genes in vivo. It has already beenshown that short anbsense oligonucleotides can be imported into cellswhere they act as inhibitors, despite their low intracellularconcentrations caused by their restricted uptake by the cell membrane.[Zamecnik et al., Proc. Natl. Acad. Sci. U.S.A. 83:4143-4146 (1986)].The oligonucleotides can be modified to enhance their uptake, e.g. bysubstituting their negatively charged phosphodiester groups by unchargedgroups.

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. The currently preferred in vivogene transfer techniques include transfection with viral (typicallyretroviral) vectors and viral coat protein-liposome mediatedtransfection [Dzau et al., Trends in Biotechnology 11:205-210 (1993)].In some situations it is desirable to provide the nucleic acid sourcewith an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262:4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. U.S.A. 87:3410-3414 (1990). For review of gene marking andgene therapy protocols see Anderson et al., Science 256:808-813 (1992).

In a preferred embodiment, IbA genes are administered as DNA vaccines,either single genes or combinations of IbA genes. Naked DNA vaccines aregenerally known in the art. Brower, Nature Biotechnology, 16:1304-1305(1998). Methods for the use of genes as DNA vaccines are well known toone of ordinary skill in the art, and include placing an IbA gene orportion of an IbA gene under the control of a promoter for expression ina patent in need of treatment. The IbA gene used for DNA vaccines canencode full-length IbA proteins, but more preferably encodes portions ofthe IbA proteins including peptides derived from the IbA protein. In apreferred embodiment a patient is immunized with a DNA vaccinecomprising a plurality of nucleotide sequences derived from an IbA gene.Similarly, it is possible to immunize a patient with a plurality of IbAgenes or portions thereof as defined herein. Without being bound bytheory, expression of the polypeptide encoded by the DNA vaccine,cytotoxic T-cells, helper T-cells and antibodies are induced whichrecognize and destroy or eliminate cells expressing IFN-β proteins.

In a preferred embodiment, the DNA vaccines include a gene encoding anadjuvant molecule with the DNA vaccine. Such adjuvant molecules includecytokines that increase the immunogenic response to the IbA polypeptideencoded by the DNA vaccine. Additional or alternative adjuvants areknown to those of ordinary skill in the art and find use in theinvention.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references cited herein are incorporated by reference in theirentirety.

EXAMPLE 1 DESIGN AND CHARACTERIZATION OF NOVEL IbA PROTEINS BY PDA

Summary: Sequences for novel interferon-beta activity proteins (IbAproteins) were designed by simultaneously optimizing residues in theburied core of the protein using Protein Design Automation (PDA) asdescribed in WO98/47089, U.S. Ser. Nos. 09/058,459, 09/127,926,60/104,612, 60/158,700, 09/419,351, 60/181,630, 60/186,904, and U.Spatent application, entitled Protein Design Automation For ProteinLibraries (Filed: Apr. 14, 2000; Inventor: Bassil Dahiyat), all of whichare expressly incorporated by reference in their entirety. Several coredesigns were completed, with 20-61 residues considered corresponding to20²⁰-20⁶¹ sequence possibilities. Residues unexposed to solvent weredesigned in order to minimize changes to the molecular surface and tolimit the potential for antigenicity of designed novel proteinanalogues.

Calculations required from 12-19 hours on 16 Silicon Graphics R10000CPU's. The global optimum sequence from each design was selected forcharacterization. From 2-11 residues were changed from human IFN-β inthe designed proteins, out of 166 residues total.

COMPUTATIONAL PROTOCOLS

Template Structure Preparation:

For this study the crystal structure of human IFN-β as deposited in thePDB data bank was used [PDB record 1AU1; Karpusasetal. Proc. Natl. Acad.Sci. U.S.A. 94(22):11813-8 (1997)]. Karpasus et al. expressed humanIFN-β in CHO cells (glycosylated form) and solved the structure by x-raycrystallography to a resolution of 2.2 Ångstrom. The structure of IFN-βis dimeric containing a zinc ion at the interface and both IFN-βmonomers (A-chain and B-chain) are glycosylated at asparagine 80.Although both monomers contained 166 amino acid residues, thecoordinates for residues 28 to 30 in the B-monomer were not given in thePDB file 1AU1. PDA calculations were performed for the A-chain andB-chain separately. The zinc ion, all water molecules and thecarbohydrate moiety as well as all hydrogen atoms that are present inthe PDB file 1AU1 were removed from the structure prior to the PDAcalculation.

Design Strategies:

Core residues were selected for design since optimization of thesepositions can improve stability, although stabilization has beenobtained from modifications at other sites as well. Core designs alsominimize changes to the molecular surface and thus limit the designedprotein's potential for antigenicity. PDA calculations were run on 3core sequences (see FIG. 3) and in a total of 15 core designs (IFN-βA-chain: Core 1, Core 2, Core 2a, Core 3, Core 4, Core 5, and Core 6;IFN-β B-chain: Core 1, Core 2, Core 2a, Core 3, Core 4, Core 5, Core 6and Core 7; see below).

PDA Calculations

All PDA calculations were performed with salvation model 2. Solvationmodel 2 is the solvation model described by Street and Mayo [Fold.Design 3:253-258 (1998)]. If possible, Dead End Elimination (DEE) wasrun to completion to find the PDA ground state. This was done for thePDA calculations for the A-chain and B-chain of Core 1, Core 2 and Core2a, as defined below. For the calculation of Core 3, Core 4, Core 5,Core 6 and Core 7, DEE was aborted after the rotamer sequence space wasreduced to less than 10²⁵ sequences. The DEE calculation was for all thegiven Core calculation followed by Monte Carlo (MC) minimization and alist of the 1000 lowest energy sequences was generated.

A similar procedure was used for the B-chain, where in a first step theside chain of Lys 33 was minimized for 50 steps followed by anadditional 50 steps of minimization of the complete B-chain structure.As the coordinates of residues 28 to 30 are missing in the B-chain, theN-terminus of Cys 31 and the C-terminus of Arg 27 were saturated with ahydrogen atom and the NH₂-group in Cys 31 and the COOH group in Arg 27were kept fixed during minimization to prevent them from moving too faraway from their initial positions.

Before the PDA calculations were started an initial preparation of thestructure was performed. For the A-chain, the side chains of Phe 50, Glu61, Lys 115, Met 117 were minimized with Biograf for 50 steps usingconjugate gradient procedure without a Coulomb potential. this isfollowed by an additional 50 steps of conjugate gradient minimizationwithout a Coulomb potential for the complete structure of the A-chainusing Biograf. This minimization procedure was chosen to remove initialbad contacts in the structure.

The PDA calculations for all the designs were run using the a2hl p0rotamer library. This library is based on the backbone-dependent rotamerlibrary of Dunbrack and Karplus (Dunbrack and Karplus, J. Mol. Biol.230(2):543-74 (1993); hereby expressly incorporated by reference) butincludes more rotamers for the aromatic and hydrophobic amino acids; X₁and X2 angle values of rotamers for all the aromatic amino acids and X₁angle values for all the other hydrophobic amino acids were expanded ±1standard deviation about the mean value reported in the Dunbrack andKarplus library. Typical PDA parameters were used: the van der Waalsscale factor was set to 0.9, the H-bond potential well-depth was set to8.0 kcal/mol, the solvation potential was calculated using type 2solvation with a nonpolar burial energy of 0.048 kcal/mol and a nonpolarexposure multiplication factor of 1.6, and the secondary structure scalefactor was set to 0.0 (secondary structure propensities were notconsidered). Calculations required from 12-24 hours on 16 SiliconGraphics R10000 CPU's.

Monte Carlo Analysis

Monte Carlo analysis of the sequences produced by PDA shows the groundstate (optimal) amino acid and amino acids allowed for each variableposition and their frequencies of occurrence (see FIGS. 4 through 29).

EXAMPLE 2 PDA Calculations for the A-chain of IFN-β

Different PDA calculations were performed for the core region of theA-chain of IFN-β. In these calculations the number of positions includedin the PDA design were varied and the effect of different PDA parameterson the resulting protein sequences, especially the ground state sequence(SEQ ID NO:4), was analyzed.

A-chain Core 1 Design

By visual inspection, the following residues were identified asbelonging to the core of the protein: Leu 6, Gln 10, Asn 14, Cys 17, Leu21, Ala 55, Ala 56, Thr 58, Ile 59, Met 62, Leu 63, Ile 66, Ile 69, Phe70, Val 84, Leu 87, Val 91, Gln 94, Leu 98, Ser 118, Leu 122, Tyr 125,Tyr 126, Ile 129, Leu 133, Ala 142, Trp 143, Val 146, Ile 150, Asn 153,Phe 154, Ile 157, and Leu 160. In the first calculation, Cys 17 was notincluded. Also excluded from the PDA design were Phe 70, Trp 143, andPhe 154, as they are known to be important in the stabilization of thecore region, and Gln 10, Thr 58, Gln 94, Ser 118 were excluded as theyform side chain H-bonds. Furthermore, residues Tyr 125, Tyr 126 and Asn153 were not considered as these amino acids are highly conserved inIFN-ps from different organisms as well as Ala 142 as its mutation toThr is known to lead to loss of function.

Thus, the following positions were included in the PDA design (see alsoFIG. 3):

 6   21  55  56  59  62  63  66  69  84  87  91 Leu Leu Ala Ala Ile MetLeu Ile Ile Val Leu Val  98 122 129 133 146 150 157 160 Leu Leu Ile LeuVal Ile Ile Leu

Met 62 was allowed to change to any PHOBIC amino acid (Ala, Val, Leu,Ile, Phe, Tyr, Trp, Met) and the other residues were allowed to changeto Ala, Val, Leu, Ile, Phe, Tyr, Trp and the PDA core solvationpotential was used including surface area calculation.

The PDA calculation resulted in the following ground state sequence (SEQID NO:4):

 6   21  55  56  59  62  63  66  69  84  87  91 Leu Leu Ala Ala Ile MetLeu Ile Ile Ile Phe Val  98 122 129 133 146 150 157 160 Leu Leu Ile LeuVal Ile Ile Leu

This sequence shows two mutations from the wild type IFN-β sequence,V84I and L87F (see FIG. 4B) (SEQ ID NO:4).

Using Monte Carlo technique a list of low energy sequences wasgenerated. The analysis of the lowest 1000 protein sequences generatedby Monte Carlo leads to the mutation pattern shown in FIG. 4A. Thus, anyprotein sequence showing mutations at the positions according to FIG. 4Awill potentially generate a more stable and active IbA. In particularthose protein sequences found among the list of the lowest 101 MCgenerated sequences (data not shown) have a high potential to result ina more stable and active IbA. A preferred IbA sequence is shown in FIG.4B (SEQ ID NO:4).

A-chain Core 2 Design

To allow more flexibility, all residues that have heavy side chain atomswithin a distance of 4 Angstrom of any heavy side chain atom of theamino acids used in the Core 1 calculation were added to the PDAcalculation. Thus, Met 1, Gln 10, Asn 14, Cys 17, Phe 38, Phe 50, Thr58, Glu 61, Phe 70, Glu 81, Gln 94, Ile 95, Leu 102, Lys 115, Tyr 125,Tyr 126, Leu 130, Tyr 138, Thr 144, Arg 147, Leu 151, Asn 153, Phe 154,Arg 159, Thr 161, Tyr 163, and Leu 164 were treated as wild type, suchthat the conformation of the amino acid side chain could change but notthe identity Gln 10, Asn 14, Cys 17, Phe 38, Phe 50, Thr 58, Phe 70, Gln94, Tyr 125, Tyr 126, Thr 144, Asn 153, Phe 154, Thr 161, and Leu 164were treated with the PDA core potential for surface area calculation.Ile 95, Leu 102, Arg 147, Leu 151, and Tyr 163 were treated with the PDAboundary potential for surface area calculation. Met 1, Glu 61, Glu 81,Lys 115, Leu 130, Tyr 138, and Arg 159 were treated with the PDA surfacepotential, but no surface area was calculated.

Thus, the following positions were included in the PDA design (see alsoFIG. 3):

 1   6  10  14  17  21  38  50  55  56  58  59 Met Leu Gln Asn Cys LeuPhe Phe Ala Ala Thr Ile 61  62   63  66  69  70  81  84 87   91  94  95Glu Met Leu Ile Ile Phe Glu Val Leu Val Gln Ile  98 102 115 122 125 126129 130 133 138 144 146 Leu Leu Lys Leu Tyr Tyr Ile Leu Leu Tyr Thr Val147 150 151 153 154 157 159 160 161 163 164 Arg Ile Leu Asn Phe Ile ArgLeu Thr Tyr Leu

The PDA calculation resulted in the following ground state sequence (SEQID NO:5):

 1   6  10  14  17  21  38  50  55  56  58  59 Met Leu Gln Asn Cys LeuPhe Phe Ala Ala Thr Ile 61  62   63  66  69  70  81  84 87  91  94  95Glu Met Leu Ile Ile Phe Glu Ile Leu Ile Gln Ile  98 102 115 122 125 126129 130 133 138 144 146 Phe Leu Lys Ile Tyr Tyr Ile Leu Leu Tyr Thr Val147 150 151 153 154 157 159 160 161 163 164 Arg Ile Leu Asn Phe Leu ArgLeu Thr Tyr Leu

This sequence shows five mutations from the wild type sequence, V84I,V91I, L98F, L122I, and I157L (see FIG. 5B) (SEQ ID NO:5).

Using Monte Carlo technique a list of low energy sequences wasgenerated. The analysis of the lowest 1000 protein sequences generatedby Monte Carlo leads to the mutation pattern shown in FIG. 5A. Thus, anyprotein sequence showing mutations at the positions according to FIG. 5Awill potentially generate a more stable and active IbA. In particularthose protein sequences found among the list of the lowest 101 MCgenerated sequences (data not shown) have a high potential to result ina more stable and active IbA. A preferred IbA sequence is shown in FIG.5B (SEQ ID NO:5).

A-chain Core 2a Design

A calculation similar to Core 2 was performed but now all wild typeresidues were treated with the PDA core potential including the surfacearea calculation. This calculation yields the same ground state sequence(SEQ ID NO:5) as resulted from Core 2.

 1   6  10  14  17  21  38  50  55  56  58  59 Met Leu Gln Asn Cys LeuPhe Phe Ala Ala Thr Ile 61  62   63  66  69  70  81  84 87   91  94  95Glu Met Leu Ile Ile Phe Glu Ile Leu Ile Gln Ile  98 102 115 122 125 126129 130 133 138 144 146 Phe Leu Lys Ile Tyr Tyr Ile Leu Leu Tyr Thr Val147 150 151 153 154 157 159 160 161 163 164 Arg Ile Leu Asn Phe Leu ArgLeu Thr Tyr Leu

A-chain Core 3 Design

A slightly larger core region than that used in core 2 was defined. Theresidues Ser 13, Cys 17, Gly 114, Ser 118, Ala 142, Trp 143, Phe 154,and Thr 161 were added to the PDA design used in core 2a and allowed tochange their identity. Ser 13, Ala 142, Trp 143, Phe 154 and Thr 161could change to any PHOBIC residues except methionine; Cys 17 to anyPHOBIC residue plus cysteine, but not to methionine; Gly 114 couldbecome any PHOBIC residue plus glycine, but not methionine; Ser 118could become any PHOBIC residue plus serine, but no methionine. Allthese eight were treated with the PDA core potential for surface areacalculation. In addition, the following residues were added and treatedas wild type using the PDA core potential for surface area calculation:Gln 18, Gln 72, Ser 74, Ser 76, Thr 77, Asn 90, Tyr 132, Lys 136, andSer 139.

Thus, the following positions were included in the PDA design (see alsoFIG. 3):

 1   6  10  13  14  17  18  21  38  50  55  56 Met Leu Gln Ser Asn CysGln Leu Phe Phe Ala Ala 58  59   61  62  63  66  69  70  72  74  76  77Thr Ile Glu Met Leu Ile Ile Phe Gln Ser Ser Thr 81  84  87  90  91  94  95  98 102 114 115 118 Glu Val Leu Asn Val GlnIle Leu Leu Gly Lys Ser 122 125 126 129 130 132 133 136 138 139 142 143Leu Tyr Tyr Ile Leu Tyr Leu Lys Tyr Ser Ala Trp 144 146 147 150 151 153154 157 159 160 161 163 Thr Val Arg Ile Leu Asn Phe Ile Arg Leu Thr Tyr164 Leu

The PDA calculation resulted in the following ground state sequence (SEQID NO:6):

 1   6  10  13  14  17  18  21  38  50  55  56 Met Leu Gln Phe Asn CysGln Leu Phe Phe Ala Ala 58  59   61  62  63  66  69  70  72  74  76  77Thr Ile Glu Met Leu Ile Val Phe Gln Ser Ser Thr 81  84  87  90  91  94  95  98 102 114 115 118 Glu Ile Leu Asn Ile GlnIle Phe Leu Gly Lys Ala 122 125 126 129 130 132 133 136 138 139 142 143Ile Tyr Tyr Ile Leu Tyr Leu Lys Tyr Ser Ala Trp 144 146 147 150 151 153154 157 159 160 161 163 Thr Ile Arg Ile Leu Asn Phe Leu Arg Leu Ala Tyr164 Leu

This sequence shows 10 mutations from the wild type sequence, S13F,I69V, V84I, V91I, L98F, S118A, L122I, V146I, I157L, and T161A (see FIG.6B) (SEQ ID NO:6).

Using Monte Carlo technique a list of low energy sequences wasgenerated. The analysis of the lowest 1000 protein sequences generatedby Monte Carlo leads to the mutation pattern shown in FIG. 6A. Thus, anyprotein sequence showing mutations at the positions according to FIG. 6Awill potentially generate a more stable and active IbA. In particularthose protein sequences found among the list of the lowest 101 MCgenerated sequences (data not shown) have a high potential to result ina more stable and active IbA. Preferred IbA sequences are shown in FIGS.6B, 6C, and 6D (SEQ ID NOS: 6-8).

A-chain Core 4 Design

The newly added residues Ser 13, Cys 17, Ser 118, and Thr 161 were nowallowed to change to any of the following amino acids: Ala, Val, Leu,Ile, Phe, Tyr, Trp, Asp, Asn, Glu, Gln, Lys, Ser, Thr, His, and Arg, butthey were still treated with the PDA core potential for surface areacalculation. Otherwise this calculation is identical to Core 3.

The PDA calculation resulted in the following ground state sequence (SEQID NO:9):

 1   6  10  13  14  17  18  21  38  50  55  56 Met Leu Gln Phe Asn AspGln Leu Phe Phe Ala Ala 58  59   61  62  63  66  69  70  72   74  76  77Thr Ile Glu Met Leu Ile Val Phe Gln Ser Ser Thr 81  84  87  90  91  94  95  98 102 114 115 118 Glu Ile Leu Asn Ile GlnIle Phe Leu Gly Lys Ala 122 125 126 129 130 132 133 136 138 139 142 143Ile Tyr Tyr Ile Leu Tyr Leu Lys Tyr Ser Ala Trp 144 146 147 150 151 153154 157 159 160 161 163 Thr Ile Arg Ile Leu Asn Phe Leu Arg Leu Ala Tyr164 Leu

This sequence shows 10 mutations from the wild type sequence, S13E,C17D, 169V, V84I, V91I, S118A, L122I, V146I, I157L, and T161A (see FIG.9B) (SEQ ID NO:15).

Using Monte Carlo technique a list of low energy sequences wasgenerated. The analysis of the lowest 1000 protein sequences generatedby Monte Carlo leads to the mutation pattern shown in FIG. 9A. Thus, anyprotein sequence showing mutations at the positions according to FIG. 9Awill potentially generate a more stable and active IbA. In particularthose protein sequences found among the list of the lowest 101 MCgenerated sequences (data not shown) have a high potential to result ina more stable and active IbA. Preferred IbA sequences are shown in FIGS.9B, 9C, and 9D (SEQ ID NOS:15-17). A DNA library can be generated tomirror the probability table of FIG. 9A that comprises at least onesequence that is more stable and/or active than wild type IFN-β.

EXAMPLE 3 PDA Calculations for the B-chain of IFN-β

For the B-chain, PDA calculations similar to those of the A-chain wereperformed.

B-chain Core 1 Design

The same positions as for the A-chain Core 1 calculation were used inthe PDA design for the B-chain: Leu 6, Leu 21, Ala 55, Ala 56, Ile 59,Met 62, Leu 63, Ile 66, Ile 69, Val 84, Leu 98, Leu 122, Ile 129, Leu133, Val 146, Ile 150, Ile 157, and Leu 160.

The PDA calculation resulted in the following ground state sequence (SEQID NO:1 8):

 6   21  55  56  59  62  63  66  69  84  87  91 Leu Leu Ala Ala IIe MetLeu Ile Ile Ile Phe Val  98 122 129 133 146 150 157 160 Leu Leu Ile LeuVal Ile Ile Leu

This sequence shows two mutations from the wild type IFN-β sequence,V84I and L87F, and is identical with the ground state sequence generatedfor the A-chain (see FIG. 10B) (SEQ ID NO:18).

Using Monte Carlo technique a list of low energy sequences wasgenerated. The analysis of the lowest 1000 protein sequences generatedby Monte Carlo leads to the mutation pattern shown in FIG. 10A. Thus,any protein sequence showing mutations at the positions according toFIG. 10A will potentially generate a more stable and active IbA. Inparticular those protein sequences found among the list of the lowest101 MC generated sequences (data not shown) have a high potential toresult in a more stable and active IbA. A preferred IbA sequence isshown in FIG. 10B (SEQ ID NO:18). A DNA library can be generated tomirror the probability table of FIG. 10A that comprises at least onesequence that is more stable and/or active than wild type IFN-β.

B-chain Core 2 Design

A calculation similar to that for the A-chain Core 2 design wasperformed for the B-chain.

The PDA calculation resulted in the following ground state sequence (SEQID NO:19):

 1   6  10  14  17  21  38  50  55  56  58  59 Met Leu Gln Asn Cys LeuPhe Phe Ala Leu Thr Ile 61  62   63  66  69  70  81  84 87  91  94  95Glu Met Phe Ile Ile Phe Glu Ile Phe Ile Gln Ile  98 102 115 122 125 126129 130 133 138 144 146 Leu Leu Lys Phe Tyr Tyr Ile Leu Leu Tyr Thr Val147 150 151 153 154 157 159 160 161 163 164 Arg Ile Leu Asn Phe Ile ArgLeu Thr Tyr Leu

This sequence shows six mutations from the wild type sequence, A56L,L63F, V84I, L87F, V91I, and L122F (see FIG. 11B) (SEQ ID NO:19).

Using Monte Carlo technique a list of low energy sequences wasgenerated. The analysis of the lowest 1000 protein sequences generatedby Monte Carlo leads to the mutation pattern shown in FIG. 11A. Thus,any protein sequence showing mutations at the positions according toFIG. 11A will potentially generate a more stable and active IbA. Inparticular those protein sequences found among the list of the lowest101 MC generated sequences (data not shown) have a high potential toresult in a more stable and active IbA. A preferred IbA sequence isshown in FIG. 11B (SEQ ID NO:19). A DNA library can be generated tomirror the probability table of FIG. 11A that comprises at least onesequence that is more stable and/or active than wild type IFN-β.

B-chain Core 2a Design

A calculation similar to that for the A-chain Core 2a design wasperformed for the B-chain. This calculation yields the same ground statesequence (SEQ ID NO:i9) as resulted from Core 2.

 1   6  10  14  17  21  38  50  55  56  58  59 Met Leu Gln Asn Cys LeuPhe Phe Ala Ala Thr Ile 61  62   63  66  69  70  81  84 87  91  94  95Glu Met Leu Ile Ile Phe Glu Ile Phe Ile Gln Ile  98 102 115 122 125 126129 130 133 138 144 146 Leu Leu Lys Phe Tyr Tyr Ile Leu Leu Tyr Thr Val147 150 151 153 154 157 159 160 161 163 164 Arg Ile Leu Asn Phe Ile ArgLeu Thr Tyr Leu

This sequence shows six mutations from the wild type sequence, A56L,L63F, V84I, L87F, V91I, and L122F.

B-chain Core 3 Design

A calculation similar to that for the A-chain Core 3 was performed forthe B-chain, but instead of residue Gln 18, Phe15 was included in thewild type PDA residue list.

The PDA calculation resulted in the following ground state sequence (SEQID NO:20):

 1   6  10  13  14  15  17  21  38  50  55  56 Met Leu Gln Leu Asn PheCys Leu Phe Phe Ala Leu 58  59   61  62  63  66  69  70  72  74  76  77Thr Ile Glu Met Leu Ile Ile Phe Gln Ser Ser Thr 81  84  87  90  91  94  95  98 102 114 115 118 Glu Ile Leu Asn Ile GlnIle Leu Leu Phe Lys Leu 122 125 126 129 130 132 133 136 138 139 142 143Ile Tyr Tyr Ile Leu Tyr Leu Lys Tyr Ser Ala Trp 144 146 147 150 151 153154 157 159 160 161 163 Thr Val Arg Ile Leu Asn Phe Ile Arg Leu Ala Tyr164 Leu

This sequence shows 8 mutations from the wild type sequence, S13L, A56L,V84I, V91I, G114F, S118L, L122I, and T161A (see FIG. 12B) (SEQ IDNO:20).

Using Monte Carlo technique a list of low energy sequences wasgenerated. The analysis of the lowest 1000 protein sequences generatedby Monte Carlo leads to the mutation pattern shown in FIG. 12A. Thus,any protein sequence showing mutations at the positions according toFIG. 12A will potentially generate a more stable and active IbA. Inparticular those protein sequences found among the list of the lowest101 MC generated sequences (data not shown) have a high potential toresult in a more stable and active IbA. A preferred IbA sequence isshown in FIG. 12B (SEQ ID NO:20). A DNA library can be generated tomirror the probability table of FIG. 12A that comprises at least onesequence that is more stable and/or active than wild type IFN-β.

B-chain Core 4 Design

A calculation similar to that for the A-chain Core 4 design wasperformed for the B-chain, but instead of residue Gln 18, Phe 15 wasincluded in the wild type PDA residue list.

The PDA calculation resulted in the following ground state sequence (SEQID NO:21):

 1   6  10  13  14  15  17  21  38  50  55  56 Met Leu Gln Leu Asn PheAla Leu Phe Phe Ala Leu 58  59   61  62  63  66  69  70  72  74  76  77Thr Ile Glu Met Leu Ile Ile Phe Gln Ser Ser Thr 81  84  87  90  91  94  95  98 102 114 115 118 Glu Ile Phe Asn Leu GlnIle Leu Leu Phe Lys Leu 122 125 126 129 130 132 133 136 138 139 142 143Ile Tyr Tyr Ile Leu Tyr Leu Lys Tyr Ser Ala Trp 144 146 147 150 151 153154 157 159 160 161 163 Thr Val Arg Ile Leu Asn Phe Ile Arg Leu Glu Tyr164 Leu

This sequence shows 10 mutations from the wild type sequence, S13L,C17A, A56L, V84I, L87F, V91L, G114F, S118L, L122I, and T161E (see FIG.13B) (SEQ ID NO:21).

Using Monte Carlo technique a list of low energy sequences wasgenerated. The analysis of the lowest 1000 protein sequences generatedby Monte Carlo leads to the mutation pattern shown in FIG. 13A. Thus,any protein sequence showing mutations at the positions according toFIG. 13A will potentially generate a more stable and active IbA. Inparticular those protein sequences found among the list of the lowest101 MC generated sequences (data not shown) have a high potential toresult in a more stable and active IbA. A preferred IbA sequence isshown in FIG. 13B (SEQ ID NO:21). A DNA library can be generated tomirror the probability table of FIG. 13A that comprises at least onesequence that is more stable and/or active than wild type IFN-β.

B-chain Core 5 Design

A calculation similar to that for the A-chain Core 5 design wasperformed for the B-chain. Now, Gln 18 was included in the wild type PDAresidue list, exactly as was done in the Core 5 calculation for theA-chain.

The PDA calculation resulted in the following ground state sequence (SEQID NO:22):

 1   6  10  13  14  17  18  21  38  50  55  56 Met Leu Gln Glu Asn CysGln Leu Phe Phe Ala Leu 58  59   61  62  63  66  69  70 72   74  76  77Thr Ile Glu Met Leu Ile Ile Phe Gln Ser Ser Thr 81  84  87  90  91  94  95  98 102 114 115 118 Glu Ile Leu Asn Ile GlnIle Leu Leu Leu Lys Glu 122 125 126 129 130 132 133 136 138 139 142 143Leu Tyr Tyr Ile Leu Tyr Leu Lys Tyr Ser Ala Trp 144 146 147 150 151 153154 157 159 160 161 163 Thr Val Arg Ile Leu Asn Phe Ile Arg Leu Glu Tyr164 Leu

This sequence shows 7 mutations from the wild type sequence, S13E, A56L,V84I, V91I, G114L, S118E, and T161E (see FIG. 14B) (SEQ ID NO:22).

Using Monte Carlo technique a list of low energy sequences wasgenerated. The analysis of the lowest 1000 protein sequences generatedby Monte Carlo leads to the mutation pattern shown in FIG. 14A. Thus,any protein sequence showing mutations at the positions according toFIG. 14A will potentially generate a more stable and active IbA. Inparticular those protein sequences found among the list of the lowest101 MC generated sequences (data not shown) have a high potential toresult in a more stable and active IbA. A preferred IbA sequence isshown in FIG. 14B (SEQ ID NO:22). A DNA library can be generated tomirror the probability table of FIG. 14A that comprises at least onesequence that is more stable and/or active than wild type IFN-β.

B-chain Core 6 Design

A similar calculation similar to that for the A-chain Core 6 design wasperformed for the B-chain.

The PDA calculation resulted in the following ground state sequence (SEQID NO:23):

 1   6  10  13  14  17  18  21  38  50  55  56 Met Leu Gln Ser Asn ThrGln Leu Phe Phe Ala Leu 58  59   61  62  63  66  69  70 72   74  76  77Thr Ile Glu Met Leu Ile Ile Phe Gln Ser Ser Thr 81  84  87  90  91  94  95  98 102 114 115 118 Glu Ile Leu Asn Ile GlnIle Leu Leu Leu Lys Glu 122 125 126 129 130 132 133 136 138 139 142 143Leu Tyr Tyr Ile Leu Tyr Leu Lys Tyr Ser Ala Trp 144 146 147 150 151 153154 157 159 160 161 163 Thr Val Arg Ile Leu Asn Phe Ile Arg Leu Glu Tyr164 Leu

This sequence shows 7 mutations from the wild type sequence, C17T, A56L,V84I, V91I, G114L, S118E, and T161E (see FIG. 15B) (SEQ ID NO:23).

Using Monte Carlo technique a list of low energy sequences wasgenerated. The analysis of the lowest 1000 protein sequences generatedby Monte Carlo leads to the mutation pattern shown in FIG. 15A. Thus,any protein sequence showing mutations at the positions according toFIG. 15A will potentially generate a more stable and active IbA. Inparticular those protein sequences found among the list of the lowest101 MC generated sequences (data not shown) have a high potential toresult in a more stable and active IbA. A preferred IbA sequence isshown in FIG. 15B (SEQ ID NO:23). A DNA library can be generated tomirror the probability table of FIG. 15A that comprises at least onesequence that is more stable and/or active than wild type IFN-β.

B-chain Core 7 Design

A similar calculation similar to that of the B-chain Core 6 design wasperformed. Now Gly 114 is treated as a wild type residue.

The PDA calculation resulted in the following ground state sequence (SEQID NO:24):

 1   6  10  13  14  17  18  21  38  50  55  56 Met Leu Gln Ser Asn ThrGln Leu Phe Phe Ala Leu 58  59   61  62  63  66  69  70 72   74  76  77Thr Ile Glu Met Leu Ile Ile Phe Gln Ser Ser Thr 81  84  87  90  91  94  95  98 102 114 115 118 Glu Ile Leu Asn Ile GlnIle Leu Leu Gly Lys Glu 122 125 126 129 130 132 133 136 138 139 142 143Leu Tyr Tyr Ile Leu Tyr Leu Lys Tyr Ser Ala Trp 144 146 147 150 151 153154 157 159 160 161 163 Thr Val Arg Ile Leu Asn Phe Ile Arg Leu Glu Tyr164 Leu

This sequence shows 6 mutations from the wild type sequence, C17T, A56L,V84I, V91I, S118E, and T161E (see FIG. 16B) (SEQ ID NO:24). With theexception of position 114, now remaining glycine, the ground statesequence is identical to that of Core 6 for the B-chain.

Using Monte Carlo technique a list of low energy sequences wasgenerated. The analysis of the lowest 1000 protein sequences generatedby Monte Carlo leads to the mutation pattern shown in FIG. 16A. Thus,any protein sequence showing mutations at the positions according toFIG. 16A will potentially generate a more stable and active IbA. Inparticular those protein sequences found among the list of the lowest101 MC generated sequences (data not shown) have a high potential toresult in a more stable and active IbA. A preferred IbA sequence isshown in FIG. 16B (SEQ ID NO:24). A DNA library can be generated tomirror the probability table of FIG. 16A that comprises at least onesequence that is more stable and/or active than wild type IFN-β.

24 1 166 PRT Homo sapiens 1 Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln ArgSer Ser Asn Phe Gln 1 5 10 15 Cys Gln Lys Leu Leu Trp Gln Leu Asn GlyArg Leu Glu Tyr Cys Leu 20 25 30 Lys Asp Arg Met Asn Phe Asp Ile Pro GluGlu Ile Lys Gln Leu Gln 35 40 45 Gln Phe Gln Lys Glu Asp Ala Ala Leu ThrIle Tyr Glu Met Leu Gln 50 55 60 Asn Ile Phe Ala Ile Phe Arg Gln Asp SerSer Ser Thr Gly Trp Asn 65 70 75 80 Glu Thr Ile Val Glu Asn Leu Leu AlaAsn Val Tyr His Gln Ile Asn 85 90 95 His Leu Lys Thr Val Leu Glu Glu LysLeu Glu Lys Glu Asp Phe Thr 100 105 110 Arg Gly Lys Leu Met Ser Ser LeuHis Leu Lys Arg Tyr Tyr Gly Arg 115 120 125 Ile Leu His Tyr Leu Lys AlaLys Glu Tyr Ser His Cys Ala Trp Thr 130 135 140 Ile Val Arg Val Glu IleLeu Arg Asn Phe Tyr Phe Ile Asn Arg Leu 145 150 155 160 Thr Gly Tyr LeuArg Asn 165 2 757 DNA Homo sapiens 2 atgaccaaca agtgtctcct ccaaattgctctcctgttgt gcttctccac tacagctctt 60 tccatgagct acaacttgct tggattcctacaaagaagca gcaattttca gtgtcagaag 120 ctcctgtggc aattgaatgg gaggcttgaatattgcctca aggacaggat gaactttgac 180 atccctgagg agattaagca gctgcagcagttccagaagg aggacgccgc attgaccatc 240 tatgagatgc tccagaacat ctttgctattttcagacaag attcatctag cactggctgg 300 aatgagacta ttgttgagaa cctcctggctaatgtctatc atcagataaa ccatctgaag 360 acagtcctgg aagaaaaact ggagaaagaagattttacca ggggaaaact catgagcagt 420 ctgcacctga aaagatatta tgggaggattctgcattacc tgaaggccaa ggagtacagt 480 cactgtgcct ggaccatagt cagagtggaaatcctaagga acttttactt cattaacaga 540 cttacaggtt acctccgaaa ctgaagatctcctagcctgt ccctctggga ctggacaatt 600 gcttcaagca ttcttcaacc agcagatgctgtttaagtga ctgatggcta atgtactgca 660 aatgaaagga cactagaaga ttttgaaatttttattaaat tatgagttat ttttatttat 720 ttaaatttta ttttggaaaa taaattatttttggtgc 757 3 21 PRT Homo sapiens 3 Met Thr Asn Lys Cys Leu Leu Gln IleAla Leu Leu Leu Cys Phe Ser 1 5 10 15 Thr Thr Ala Leu Ser 20 4 166 PRTArtificial Sequence synthetic 4 Met Ser Tyr Asn Leu Leu Gly Phe Leu GlnArg Ser Ser Asn Phe Gln 1 5 10 15 Cys Gln Lys Leu Leu Trp Gln Leu AsnGly Arg Leu Glu Tyr Cys Leu 20 25 30 Lys Asp Arg Met Asn Phe Asp Ile ProGlu Glu Ile Lys Gln Leu Gln 35 40 45 Gln Phe Gln Lys Glu Asp Ala Ala LeuThr Ile Tyr Glu Met Leu Gln 50 55 60 Asn Ile Phe Ala Ile Phe Arg Gln AspSer Ser Ser Thr Gly Trp Asn 65 70 75 80 Glu Thr Ile Ile Glu Asn Phe LeuAla Asn Val Tyr His Gln Ile Asn 85 90 95 His Leu Lys Thr Val Leu Glu GluLys Leu Glu Lys Glu Asp Phe Thr 100 105 110 Arg Gly Lys Leu Met Ser SerLeu His Leu Lys Arg Tyr Tyr Gly Arg 115 120 125 Ile Leu His Tyr Leu LysAla Lys Glu Tyr Ser His Cys Ala Trp Thr 130 135 140 Ile Val Arg Val GluIle Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu 145 150 155 160 Thr Gly TyrLeu Arg Asn 165 5 166 PRT Artificial Sequence synthetic 5 Met Ser TyrAsn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln 1 5 10 15 Cys GlnLys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu 20 25 30 Lys AspArg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln 35 40 45 Gln PheGln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln 50 55 60 Asn IlePhe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn 65 70 75 80 GluThr Ile Ile Glu Asn Leu Leu Ala Asn Ile Tyr His Gln Ile Asn 85 90 95 HisPhe Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr 100 105 110Arg Gly Lys Leu Met Ser Ser Leu His Ile Lys Arg Tyr Tyr Gly Arg 115 120125 Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr 130135 140 Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Leu Asn Arg Leu145 150 155 160 Thr Gly Tyr Leu Arg Asn 165 6 166 PRT ArtificialSequence synthetic 6 Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser PheAsn Phe Gln 1 5 10 15 Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg LeuGlu Tyr Cys Leu 20 25 30 Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu IleLys Gln Leu Gln 35 40 45 Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile TyrGlu Met Leu Gln 50 55 60 Asn Ile Phe Ala Val Phe Arg Gln Asp Ser Ser SerThr Gly Trp Asn 65 70 75 80 Glu Thr Ile Ile Glu Asn Leu Leu Ala Asn IleTyr His Gln Ile Asn 85 90 95 His Phe Lys Thr Val Leu Glu Glu Lys Leu GluLys Glu Asp Phe Thr 100 105 110 Arg Gly Lys Leu Met Ala Ser Leu His IleLys Arg Tyr Tyr Gly Arg 115 120 125 Ile Leu His Tyr Leu Lys Ala Lys GluTyr Ser His Cys Ala Trp Thr 130 135 140 Ile Ile Arg Val Glu Ile Leu ArgAsn Phe Tyr Phe Leu Asn Arg Leu 145 150 155 160 Ala Gly Tyr Leu Arg Asn165 7 166 PRT Artificial Sequence synthetic 7 Met Ser Tyr Asn Leu LeuGly Phe Leu Gln Arg Ser Tyr Asn Phe Gln 1 5 10 15 Cys Gln Lys Leu LeuTrp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu 20 25 30 Lys Asp Arg Met AsnPhe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln 35 40 45 Gln Phe Gln Lys GluAsp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln 50 55 60 Asn Ile Phe Ala ValPhe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn 65 70 75 80 Glu Thr Ile IleGlu Asn Leu Leu Ala Asn Ile Tyr His Gln Ile Asn 85 90 95 His Phe Lys ThrVal Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr 100 105 110 Arg Gly LysLeu Met Val Ser Leu His Val Lys Arg Tyr Tyr Gly Arg 115 120 125 Ile LeuHis Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr 130 135 140 IleIle Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Leu Asn Arg Leu 145 150 155160 Ala Gly Tyr Leu Arg Asn 165 8 166 PRT Artificial Sequence synthetic8 Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Phe Asn Phe Gln 1 5 1015 Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu 20 2530 Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln 35 4045 Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln 50 5560 Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn 65 7075 80 Glu Thr Ile Ile Glu Asn Leu Leu Ala Asn Ile Tyr His Gln Ile Asn 8590 95 His Phe Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr100 105 110 Arg Gly Lys Leu Met Ala Ser Leu His Ile Lys Arg Tyr Tyr GlyArg 115 120 125 Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys AlaTrp Thr 130 135 140 Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe LeuAsn Arg Leu 145 150 155 160 Ala Gly Tyr Leu Arg Asn 165 9 166 PRTArtificial Sequence synthetic 9 Met Ser Tyr Asn Leu Leu Gly Phe Leu GlnArg Ser Phe Asn Phe Gln 1 5 10 15 Asp Gln Lys Leu Leu Trp Gln Leu AsnGly Arg Leu Glu Tyr Cys Leu 20 25 30 Lys Asp Arg Met Asn Phe Asp Ile ProGlu Glu Ile Lys Gln Leu Gln 35 40 45 Gln Phe Gln Lys Glu Asp Ala Ala LeuThr Ile Tyr Glu Met Leu Gln 50 55 60 Asn Ile Phe Ala Val Phe Arg Gln AspSer Ser Ser Thr Gly Trp Asn 65 70 75 80 Glu Thr Ile Ile Glu Asn Leu LeuAla Asn Ile Tyr His Gln Ile Asn 85 90 95 His Phe Lys Thr Val Leu Glu GluLys Leu Glu Lys Glu Asp Phe Thr 100 105 110 Arg Gly Lys Leu Met Ala SerLeu His Ile Lys Arg Tyr Tyr Gly Arg 115 120 125 Ile Leu His Tyr Leu LysAla Lys Glu Tyr Ser His Cys Ala Trp Thr 130 135 140 Ile Ile Arg Val GluIle Leu Arg Asn Phe Tyr Phe Leu Asn Arg Leu 145 150 155 160 Ala Gly TyrLeu Arg Asn 165 10 166 PRT Artificial Sequence synthetic 10 Met Ser TyrAsn Leu Leu Gly Phe Leu Gln Arg Ser Tyr Asn Phe Gln 1 5 10 15 Asp GlnLys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu 20 25 30 Lys AspArg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln 35 40 45 Gln PheGln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln 50 55 60 Asn IlePhe Ala Val Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn 65 70 75 80 GluThr Ile Ile Glu Asn Leu Leu Ala Asn Ile Tyr His Gln Ile Asn 85 90 95 HisPhe Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr 100 105 110Arg Gly Lys Leu Met Val Ser Leu His Val Lys Arg Tyr Tyr Gly Arg 115 120125 Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr 130135 140 Ile Ile Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Leu Asn Arg Leu145 150 155 160 Ala Gly Tyr Leu Arg Asn 165 11 166 PRT ArtificialSequence synthetic 11 Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg SerPhe Asn Phe Gln 1 5 10 15 Asp Gln Lys Leu Leu Trp Gln Leu Asn Gly ArgLeu Glu Tyr Cys Leu 20 25 30 Lys Asp Arg Met Asn Phe Asp Ile Pro Glu GluIle Lys Gln Leu Gln 35 40 45 Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr IleTyr Glu Met Leu Gln 50 55 60 Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser SerSer Thr Gly Trp Asn 65 70 75 80 Glu Thr Ile Ile Glu Asn Leu Leu Ala AsnIle Tyr His Gln Ile Asn 85 90 95 His Phe Lys Thr Val Leu Glu Glu Lys LeuGlu Lys Glu Asp Phe Thr 100 105 110 Arg Gly Lys Leu Met Ala Ser Leu HisIle Lys Arg Tyr Tyr Gly Arg 115 120 125 Ile Leu His Tyr Leu Lys Ala LysGlu Tyr Ser His Cys Ala Trp Thr 130 135 140 Ile Val Arg Val Glu Ile LeuArg Asn Phe Tyr Phe Leu Asn Arg Leu 145 150 155 160 Ala Gly Tyr Leu ArgAsn 165 12 166 PRT Artificial Sequence synthetic 12 Met Ser Tyr Asn LeuLeu Gly Phe Leu Gln Arg Ser Glu Asn Phe Gln 1 5 10 15 Asp Gln Lys LeuLeu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu 20 25 30 Lys Asp Arg MetAsn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln 35 40 45 Gln Phe Gln LysGlu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln 50 55 60 Asn Ile Phe AlaIle Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn 65 70 75 80 Glu Thr IleIle Glu Asn Leu Leu Ala Asn Ile Tyr His Gln Ile Asn 85 90 95 His Leu LysThr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr 100 105 110 Arg GlyLys Leu Met Cys Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg 115 120 125 IleLeu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr 130 135 140Ile Ile Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu 145 150155 160 Cys Gly Tyr Leu Arg Asn 165 13 166 PRT Artificial Sequencesynthetic 13 Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ala Asn PheGln 1 5 10 15 Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu TyrCys Leu 20 25 30 Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys GlnLeu Gln 35 40 45 Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu MetLeu Gln 50 55 60 Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr GlyTrp Asn 65 70 75 80 Glu Thr Ile Ile Glu Asn Leu Leu Ala Asn Ile Tyr HisGln Ile Asn 85 90 95 His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys GluAsp Phe Thr 100 105 110 Arg Gly Lys Leu Met Cys Ser Leu His Leu Lys ArgTyr Tyr Gly Arg 115 120 125 Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr SerHis Cys Ala Trp Thr 130 135 140 Ile Ile Arg Val Glu Ile Leu Arg Asn PheTyr Phe Leu Asn Arg Leu 145 150 155 160 Cys Gly Tyr Leu Arg Asn 165 14166 PRT Artificial Sequence synthetic 14 Met Ser Tyr Asn Leu Leu Gly PheLeu Gln Arg Ser Glu Asn Phe Gln 1 5 10 15 Asp Gln Lys Leu Leu Trp GlnLeu Asn Gly Arg Leu Glu Tyr Cys Leu 20 25 30 Lys Asp Arg Met Asn Phe AspIle Pro Glu Glu Ile Lys Gln Leu Gln 35 40 45 Gln Phe Gln Lys Glu Asp AlaAla Leu Thr Ile Tyr Glu Met Leu Gln 50 55 60 Asn Ile Phe Ala Ile Phe ArgGln Asp Ser Ser Ser Thr Gly Trp Asn 65 70 75 80 Glu Thr Ile Ile Glu AsnLeu Leu Ala Asn Ile Tyr His Gln Ile Asn 85 90 95 His Leu Lys Thr Val LeuGlu Glu Lys Leu Glu Lys Glu Asp Phe Thr 100 105 110 Arg Gly Lys Leu MetCys Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg 115 120 125 Ile Leu His TyrLeu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr 130 135 140 Ile Val ArgVal Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu 145 150 155 160 CysGly Tyr Leu Arg Asn 165 15 166 PRT Artificial Sequence synthetic 15 MetSer Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Glu Asn Phe Gln 1 5 10 15Asp Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu 20 25 30Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln 35 40 45Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln 50 55 60Asn Ile Phe Ala Val Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn 65 70 7580 Glu Thr Ile Ile Glu Asn Leu Leu Ala Asn Ile Tyr His Gln Ile Asn 85 9095 His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr 100105 110 Arg Gly Lys Leu Met Ala Ser Leu His Ile Lys Arg Tyr Tyr Gly Arg115 120 125 Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala TrpThr 130 135 140 Ile Ile Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Leu AsnArg Leu 145 150 155 160 Ala Gly Tyr Leu Arg Asn 165 16 166 PRTArtificial Sequence synthetic 16 Met Ser Tyr Asn Leu Leu Gly Phe Leu GlnArg Ser Glu Asn Phe Gln 1 5 10 15 Asp Gln Lys Leu Leu Trp Gln Leu AsnGly Arg Leu Glu Tyr Cys Leu 20 25 30 Lys Asp Arg Met Asn Phe Asp Ile ProGlu Glu Ile Lys Gln Leu Gln 35 40 45 Gln Phe Gln Lys Glu Asp Ala Ala LeuThr Ile Tyr Glu Met Leu Gln 50 55 60 Asn Ile Phe Ala Ile Phe Arg Gln AspSer Ser Ser Thr Gly Trp Asn 65 70 75 80 Glu Thr Ile Ile Glu Asn Leu LeuAla Asn Ile Tyr His Gln Ile Asn 85 90 95 His Leu Lys Thr Val Leu Glu GluLys Leu Glu Lys Glu Asp Phe Thr 100 105 110 Arg Gly Lys Leu Met Ala SerLeu His Leu Lys Arg Tyr Tyr Gly Arg 115 120 125 Ile Leu His Tyr Leu LysAla Lys Glu Tyr Ser His Cys Ala Trp Thr 130 135 140 Ile Ile Arg Val GluIle Leu Arg Asn Phe Tyr Phe Leu Asn Arg Leu 145 150 155 160 Thr Gly TyrLeu Arg Asn 165 17 166 PRT Artificial Sequence synthetic 17 Met Ser TyrAsn Leu Leu Gly Phe Leu Gln Arg Ser Glu Asn Phe Gln 1 5 10 15 Asp GlnLys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu 20 25 30 Lys AspArg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln 35 40 45 Gln PheGln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln 50 55 60 Asn IlePhe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn 65 70 75 80 GluThr Ile Ile Glu Asn Leu Leu Ala Asn Ile Tyr His Gln Ile Asn 85 90 95 HisLeu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr 100 105 110Arg Gly Lys Leu Met Ala Ser Leu His Ile Lys Arg Tyr Tyr Gly Arg 115 120125 Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr 130135 140 Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Leu Asn Arg Leu145 150 155 160 Ala Gly Tyr Leu Arg Asn 165 18 166 PRT ArtificialSequence synthetic 18 Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg SerSer Asn Phe Gln 1 5 10 15 Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly ArgLeu Glu Tyr Cys Leu 20 25 30 Lys Asp Arg Met Asn Phe Asp Ile Pro Glu GluIle Lys Gln Leu Gln 35 40 45 Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr IleTyr Glu Met Leu Gln 50 55 60 Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser SerSer Thr Gly Trp Asn 65 70 75 80 Glu Thr Ile Ile Glu Asn Phe Leu Ala AsnVal Tyr His Gln Ile Asn 85 90 95 His Leu Lys Thr Val Leu Glu Glu Lys LeuGlu Lys Glu Asp Phe Thr 100 105 110 Arg Gly Lys Leu Met Ser Ser Leu HisLeu Lys Arg Tyr Tyr Gly Arg 115 120 125 Ile Leu His Tyr Leu Lys Ala LysGlu Tyr Ser His Cys Ala Trp Thr 130 135 140 Ile Val Arg Val Glu Ile LeuArg Asn Phe Tyr Phe Ile Asn Arg Leu 145 150 155 160 Thr Gly Tyr Leu ArgAsn 165 19 166 PRT Artificial Sequence synthetic 19 Met Ser Tyr Asn LeuLeu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln 1 5 10 15 Cys Gln Lys LeuLeu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu 20 25 30 Lys Asp Arg MetAsn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln 35 40 45 Gln Phe Gln LysGlu Asp Ala Leu Leu Thr Ile Tyr Glu Met Phe Gln 50 55 60 Asn Ile Phe AlaIle Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn 65 70 75 80 Glu Thr IleIle Glu Asn Phe Leu Ala Asn Ile Tyr His Gln Ile Asn 85 90 95 His Leu LysThr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr 100 105 110 Arg GlyLys Leu Met Ser Ser Leu His Phe Lys Arg Tyr Tyr Gly Arg 115 120 125 IleLeu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr 130 135 140Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu 145 150155 160 Thr Gly Tyr Leu Arg Asn 165 20 166 PRT Artificial Sequencesynthetic 20 Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Leu Asn PheGln 1 5 10 15 Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu TyrCys Leu 20 25 30 Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys GlnLeu Gln 35 40 45 Gln Phe Gln Lys Glu Asp Ala Leu Leu Thr Ile Tyr Glu MetLeu Gln 50 55 60 Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr GlyTrp Asn 65 70 75 80 Glu Thr Ile Ile Glu Asn Leu Leu Ala Asn Ile Tyr HisGln Ile Asn 85 90 95 His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys GluAsp Phe Thr 100 105 110 Arg Phe Lys Leu Met Leu Ser Leu His Ile Lys ArgTyr Tyr Gly Arg 115 120 125 Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr SerHis Cys Ala Trp Thr 130 135 140 Ile Val Arg Val Glu Ile Leu Arg Asn PheTyr Phe Ile Asn Arg Leu 145 150 155 160 Ala Gly Tyr Leu Arg Asn 165 21166 PRT Artificial Sequence synthetic 21 Met Ser Tyr Asn Leu Leu Gly PheLeu Gln Arg Ser Leu Asn Phe Gln 1 5 10 15 Ala Gln Lys Leu Leu Trp GlnLeu Asn Gly Arg Leu Glu Tyr Cys Leu 20 25 30 Lys Asp Arg Met Asn Phe AspIle Pro Glu Glu Ile Lys Gln Leu Gln 35 40 45 Gln Phe Gln Lys Glu Asp AlaLeu Leu Thr Ile Tyr Glu Met Leu Gln 50 55 60 Asn Ile Phe Ala Ile Phe ArgGln Asp Ser Ser Ser Thr Gly Trp Asn 65 70 75 80 Glu Thr Ile Ile Glu AsnPhe Leu Ala Asn Leu Tyr His Gln Ile Asn 85 90 95 His Leu Lys Thr Val LeuGlu Glu Lys Leu Glu Lys Glu Asp Phe Thr 100 105 110 Arg Phe Lys Leu MetLeu Ser Leu His Ile Lys Arg Tyr Tyr Gly Arg 115 120 125 Ile Leu His TyrLeu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr 130 135 140 Ile Val ArgVal Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu 145 150 155 160 GluGly Tyr Leu Arg Asn 165 22 166 PRT Artificial Sequence synthetic 22 MetSer Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Glu Asn Phe Gln 1 5 10 15Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu 20 25 30Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln 35 40 45Gln Phe Gln Lys Glu Asp Ala Leu Leu Thr Ile Tyr Glu Met Leu Gln 50 55 60Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn 65 70 7580 Glu Thr Ile Ile Glu Asn Leu Leu Ala Asn Ile Tyr His Gln Ile Asn 85 9095 His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr 100105 110 Arg Leu Lys Leu Met Glu Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg115 120 125 Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala TrpThr 130 135 140 Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile AsnArg Leu 145 150 155 160 Glu Gly Tyr Leu Arg Asn 165 23 166 PRTArtificial Sequence synthetic 23 Met Ser Tyr Asn Leu Leu Gly Phe Leu GlnArg Ser Ser Asn Phe Gln 1 5 10 15 Thr Gln Lys Leu Leu Trp Gln Leu AsnGly Arg Leu Glu Tyr Cys Leu 20 25 30 Lys Asp Arg Met Asn Phe Asp Ile ProGlu Glu Ile Lys Gln Leu Gln 35 40 45 Gln Phe Gln Lys Glu Asp Ala Leu LeuThr Ile Tyr Glu Met Leu Gln 50 55 60 Asn Ile Phe Ala Ile Phe Arg Gln AspSer Ser Ser Thr Gly Trp Asn 65 70 75 80 Glu Thr Ile Ile Glu Asn Leu LeuAla Asn Ile Tyr His Gln Ile Asn 85 90 95 His Leu Lys Thr Val Leu Glu GluLys Leu Glu Lys Glu Asp Phe Thr 100 105 110 Arg Leu Lys Leu Met Glu SerLeu His Leu Lys Arg Tyr Tyr Gly Arg 115 120 125 Ile Leu His Tyr Leu LysAla Lys Glu Tyr Ser His Cys Ala Trp Thr 130 135 140 Ile Val Arg Val GluIle Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu 145 150 155 160 Glu Gly TyrLeu Arg Asn 165 24 166 PRT Artificial Sequence synthetic 24 Met Ser TyrAsn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln 1 5 10 15 Thr GlnLys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu 20 25 30 Lys AspArg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln 35 40 45 Gln PheGln Lys Glu Asp Ala Leu Leu Thr Ile Tyr Glu Met Leu Gln 50 55 60 Asn IlePhe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn 65 70 75 80 GluThr Ile Ile Glu Asn Leu Leu Ala Asn Ile Tyr His Gln Ile Asn 85 90 95 HisLeu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr 100 105 110Arg Gly Lys Leu Met Glu Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg 115 120125 Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr 130135 140 Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu145 150 155 160 Glu Gly Tyr Leu Arg Asn 165

I claim:
 1. A non-naturally occurring interferon-beta activity (IbA)protein comprising at least fifteen amino acid substitutions as comparedto human IFN-β protein (SEQ ID NO: 1), wherein said substitutions areselected from amino acid residues at positions 6, 13, 17, 21, 56, 59,61, 62, 63, 66, 69, 84, 87, 91, 98, 102, 114, 118, 122, 129, 146, 150,154, 157, 160, and 161, wherein said protein exhibits at least 50% ofthe biological activity of human IFN-β protein.
 2. The non-naturallyoccurring IbA protein according to claim 1, wherein said amino acidsubstitutions are selected from positions 13, 17, 56, 63, 69, 84, 87,91, 98, 114, 118, 122, 146, 157, and
 161. 3. The non-naturally occurringIbA protein according to claim 2, wherein said substitutions areselected from the group of substitutions consisting of S13F, S13Y, S13E,S13A, S13L, C17D, C17A, C17T, A56L, L63F, I69V, V84I, V91I, L98F, G114F,G114L, S118L, S118E, S118A, S118V, S118C, L122I, L122F, L122V, V146I,I157L, T161A, T161E, and T161C.
 4. The non-naturally occurring IbAprotein according to claim 1 comprising substitutions at positions 13,17, 69, 84, 87, 91, 98, 118, 122, 146, 157, and
 161. 5. Thenon-naturally occurring IbA protein according to claim 4, wherein saidsubstitutions are selected from the group of substitutions consisting ofS13F, S13Y, S13E, S13A, C17D, 169V, V84I, L87F, V91I, L98F, S118A,S118V, S118C, L122I, L122F, I157L, T161A, and T161C.
 6. Thenon-naturally occurring IbA protein according to claim 1 comprisingsubstitutions at positions 13, 17, 56, 63, 84, 87, 91, 114, 118, 122,and
 161. 7. The non-naturally occurring IbA protein according to claim6, wherein said substitutions are selected from the group ofsubstitutions consisting of S13E, S13L, C17A, C17T, A56L, L63F, V84I,L87F, V91I, G114F, G114L, S118L, S118E, L122I, L122F, T161A, and T161E.8. A pharmaceutical composition comprising an IbA protein according toclaim 1 and a pharmaceutical carrier.
 9. A non-naturally occurringprotein according to claim 1 wherein said biological activity is theability to bind to an IFN receptor.
 10. A non-naturally occurringprotein according to claim 1 wherein said biological activity is theability to inhibit cell proliferation.
 11. A non-naturally occurringprotein according to claim 1 wherein said biological activity is theability to inhibit viral infections.
 12. A recombinant nucleic acidencoding the non-naturally occurring IbA protein of claim
 1. 13. Anexpression vector comprising the recombinant nucleic acid of claim 12.14. A host cell comprising the recombinant nucleic acid of claim
 12. 15.A host cell comprising the expression vector of claim
 13. 16. A methodof producing a non-naturally occurring IbA protein comprising culturingthe host cell of claim 15 under conditions suitable for expression ofsaid nucleic acid.
 17. The method according to claim 16 furthercomprising recovering said IbA protein.